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The PRY/SPRY/B30.2 Domain of Butyrophilin 1A1 (BTN1A1) Binds to Xanthine Oxidoreductase

IMPLICATIONS FOR THE FUNCTION OF BTN1A1 IN THE MAMMARY GLAND AND OTHER TISSUES*
Open AccessPublished:June 15, 2009DOI:https://doi.org/10.1074/jbc.M109.020446
      Butyrophilin 1A1 (BTN1A1) and xanthine oxidoreductase (XOR) are highly expressed in the lactating mammary gland and are secreted into milk associated with the milk fat globule membrane (MFGM). Ablation of the genes encoding either protein causes severe defects in the secretion of milk lipid droplets, suggesting that the two proteins may function in the same pathway. Therefore, we determined whether BTN1A1 and XOR directly interact using protein binding assays, surface plasmon resonance analysis, and gel filtration. Bovine XOR bound with high affinity in a pH- and salt-sensitive manner (KD = 101 ± 31 nm in 10 mm HEPES, 150 mm NaCl, pH 7.4) to the PRY/SPRY/B30.2 domain in the cytoplasmic region of bovine BTN1A1. Binding was stoichiometric, with one XOR dimer binding to either two BTN1A1 monomers or one dimer. XOR bound to BTN1A1 orthologs from mice, humans, or cows but not to the cytoplasmic domains of the closely related human paralogs, BTN2A1 or BTN3A1, or to the B30.2 domain of human RoRet (TRIM 38), a protein in the TRIM family. Analysis of the protein composition of the MFGM of wild type and BTN1A1 null mice showed that most of the XOR in mice lacking BTN1A1 was released from the MFGM in a soluble form when the milk lipid droplets were disrupted to prepare membrane, compared with wild-type mice, in which most of the XOR remained membrane-bound. Thus BTN1A1 functions in vivo to stabilize the association of XOR with the MFGM by direct interactions through the PRY/SPRY/B30.2 domain. The potential significance of BTN1A1/XOR interactions in the mammary gland and other tissues is discussed.
      Members of the butyrophilin (BTN)
      The abbreviations used are: BTN
      butyrophilin (orthologs of BTN in mice, cows, and humans are denoted mBTN, bBTN, and hBTN, respectively, and paralogs are denoted by the appropriate designation (e.g. hBTN1A1, hBTN2A1, and hBTN3A1)
      BTN1A1cyto
      cytoplasmic domain of BTN1A1
      GST
      glutathione S-transferase
      ECFP
      enhanced cyan fluorescent protein
      EYFP
      enhanced yellow fluorescent protein
      GFP
      green fluorescent protein
      bXOR and bB30.2
      bovine XOR and B30.2, respectively
      mXOR and mB30.2
      mouse XOR and B30.2, respectively
      MFGM
      milk fat globule membrane
      PBS
      phosphate-buffered saline
      SPR
      surface plasmon resonance
      TBS
      Tris-buffered saline
      XOR
      xanthine oxidoreductase
      MOG
      myelin oligodendrocyte glycoprotein
      RIPA
      radioimmune precipitation
      FPLC
      fast protein liquid chromatography
      MES
      4-morpholineethanesulfonic acid
      Tricine
      N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine.
      3The abbreviations used are: BTN
      butyrophilin (orthologs of BTN in mice, cows, and humans are denoted mBTN, bBTN, and hBTN, respectively, and paralogs are denoted by the appropriate designation (e.g. hBTN1A1, hBTN2A1, and hBTN3A1)
      BTN1A1cyto
      cytoplasmic domain of BTN1A1
      GST
      glutathione S-transferase
      ECFP
      enhanced cyan fluorescent protein
      EYFP
      enhanced yellow fluorescent protein
      GFP
      green fluorescent protein
      bXOR and bB30.2
      bovine XOR and B30.2, respectively
      mXOR and mB30.2
      mouse XOR and B30.2, respectively
      MFGM
      milk fat globule membrane
      PBS
      phosphate-buffered saline
      SPR
      surface plasmon resonance
      TBS
      Tris-buffered saline
      XOR
      xanthine oxidoreductase
      MOG
      myelin oligodendrocyte glycoprotein
      RIPA
      radioimmune precipitation
      FPLC
      fast protein liquid chromatography
      MES
      4-morpholineethanesulfonic acid
      Tricine
      N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine.
      gene family are attracting increasing attention because they may play multifunctional roles in diverse physiologies, including lactation (
      • Ogg S.L.
      • Weldon A.K.
      • Dobbie L.
      • Smith A.J.
      • Mather I.H.
      ,
      • Mather I.H.
      • Keenan T.W.
      ), selection and regulation of T-cells in the immune system (
      • Valentonyte R.
      • Hampe J.
      • Huse K.
      • Rosenstiel P.
      • Albrecht M.
      • Stenzel A.
      • Nagy M.
      • Gaede K.I.
      • Franke A.
      • Haesler R.
      • Koch A.
      • Lengauer T.
      • Seegert D.
      • Reiling N.
      • Ehlers S.
      • Schwinger E.
      • Platzer M.
      • Krawczak M.
      • Müller-Quernheim J.
      • Schürmann M.
      • Schreiber S.
      ,
      • Nguyen T.
      • Liu X.K.
      • Zhang Y.
      • Dong C.
      ,
      • Arnett H.A.
      • Escobar S.S.
      • Gonzalez-Suarez E.
      • Budelsky A.L.
      • Steffen L.A.
      • Boiani N.
      • Zhang M.
      • Siu G.
      • Brewer A.W.
      • Viney J.L.
      ,
      • Boyden L.M.
      • Lewis J.M.
      • Barbee S.D.
      • Bas A.
      • Girardi M.
      • Hayday A.C.
      • Tigelaar R.E.
      • Lifton R.P.
      ), and modulation of autoimmune disease (
      • Stefferl A.
      • Schubart A.
      • Storch M.
      • Amini A.
      • Mather I.
      • Lassmann H.
      • Linington C.
      ,
      • Guggenmos J.
      • Schubart A.S.
      • Ogg S.
      • Andersson M.
      • Olsson T.
      • Mather I.H.
      • Linington C.
      ,
      • Mañá P.
      • Goodyear M.
      • Bernard C.
      • Tomioka R.
      • Freire-Garabal M.
      • Liñares D.
      ). BTN proteins have the canonical structures of cell surface receptors, which, after an N-terminal signal sequence, generally comprise two exoplasmic Ig folds (
      • Gardinier M.V.
      • Amiguet P.
      • Linington C.
      • Matthieu J.M.
      ,
      • Linsley P.S.
      • Peach R.
      • Gladstone P.
      • Bajorath J.
      ), a membrane anchor and a cytoplasmic domain consisting of a stem region, a PRY/SPRY/B30.2 domain (
      • Vernet C.
      • Boretto J.
      • Mattéi M.G.
      • Takahashi M.
      • Jack L.J.
      • Mather I.H.
      • Rouquier S.
      • Pontarotti P.
      ,
      • Ponting C.
      • Schultz J.
      • Bork P.
      ), and a cytoplasmic tail at the C terminus (
      • Jack L.J.
      • Mather I.H.
      ).
      The eponymous BTN1A1 protein has been linked to the secretion of milk lipid droplets because it is highly expressed in the mammary epithelium during lactation and is incorporated into the surface membrane coat surrounding cytoplasmic lipid droplets (the milk fat globule membrane (MFGM)) as they bud into milk from the apical surface (
      • Franke W.W.
      • Heid H.W.
      • Grund C.
      • Winter S.
      • Freudenstein C.
      • Schmid E.
      • Jarasch E.D.
      • Keenan T.W.
      ). Furthermore, ablation of the Btn1a1 gene disrupts lipid secretion, causing the accumulation of large pools of triacylglycerol in the cytoplasm of Btn1a1 null mice (
      • Ogg S.L.
      • Weldon A.K.
      • Dobbie L.
      • Smith A.J.
      • Mather I.H.
      ). In a different context, dietary exposure to BTN1A1 in dairy products has been associated with modulation of the autoimmune disease multiple sclerosis because of structural similarities between the IgI fold of BTN1A1 (
      • Harpaz Y.
      • Chothia C.
      ) and the IgV fold of myelin oligodendrocyte glycoprotein (MOG) (
      • Breithaupt C.
      • Schubart A.
      • Zander H.
      • Skerra A.
      • Huber R.
      • Linington C.
      • Jacob U.
      ) an antigen on the myelin nerve sheath that is a target for autoantibodies in multiple sclerosis patients (
      • Guggenmos J.
      • Schubart A.S.
      • Ogg S.
      • Andersson M.
      • Olsson T.
      • Mather I.H.
      • Linington C.
      ,
      • Mañá P.
      • Goodyear M.
      • Bernard C.
      • Tomioka R.
      • Freire-Garabal M.
      • Liñares D.
      ,
      • Gardinier M.V.
      • Amiguet P.
      • Linington C.
      • Matthieu J.M.
      ).
      Potential interactions between the exoplasmic Ig folds of several BTN proteins, and putative receptors on immune cells are postulated to regulate positive selection of epidermal γδ-T cells in the case of Skint1 (
      • Boyden L.M.
      • Lewis J.M.
      • Barbee S.D.
      • Bas A.
      • Girardi M.
      • Hayday A.C.
      • Tigelaar R.E.
      • Lifton R.P.
      ) and suppress T-cell activation in the case of BTNL2 (
      • Nguyen T.
      • Liu X.K.
      • Zhang Y.
      • Dong C.
      ,
      • Arnett H.A.
      • Escobar S.S.
      • Gonzalez-Suarez E.
      • Budelsky A.L.
      • Steffen L.A.
      • Boiani N.
      • Zhang M.
      • Siu G.
      • Brewer A.W.
      • Viney J.L.
      ). In addition, BTN2A1 binds to the C-type lectin, DC-SIGN, on immature dendritic cells (
      • Malcherek G.
      • Mayr L.
      • Roda-Navarro P.
      • Rhodes D.
      • Miller N.
      • Trowsdale J.
      ), and proteins in the BTN3A1–3 subfamily bind to an unidentified ligand on various immune cells (
      • Compte E.
      • Pontarotti P.
      • Collette Y.
      • Lopez M.
      • Olive D.
      ).
      Interactions between the cytoplasmic domain of BTN and intracellular proteins have not been investigated in any detail. The intracellular region of most BTNs is dominated by the B30.2 or the PRY/SPRY domain, which comprises two sheets of antiparallel β-strands folded into a β- sandwich, which in some proteins is contiguous at the N terminus with one or two α-helices (
      • Grütter C.
      • Briand C.
      • Capitani G.
      • Mittl P.R.
      • Papin S.
      • Tschopp J.
      • Grütter M.G.
      ,
      • Woo J.S.
      • Imm J.H.
      • Min C.K.
      • Kim K.J.
      • Cha S.S.
      • Oh B.H.
      ,
      • Woo J.S.
      • Suh H.Y.
      • Park S.Y.
      • Oh B.H.
      ,
      • James L.C.
      • Keeble A.H.
      • Khan Z.
      • Rhodes D.A.
      • Trowsdale J.
      ,
      • Kuang Z.
      • Yao S.
      • Xu Y.
      • Lewis R.S.
      • Low A.
      • Masters S.L.
      • Willson T.A.
      • Kolesnik T.B.
      • Nicholson S.E.
      • Garrett T.J.
      • Norton R.S.
      ) (for a discussion of the relationship between PRY, SPRY, and B30.2 domains, see Ref. (
      • Rhodes D.A.
      • de Bono B.
      • Trowsdale J.
      )). This domain (here abbreviated as B30.2),
      The B30.2 domain was first defined as a region of similarity in linear sequence encoded by a single exon in BTN and TRIM protein genes (
      • Vernet C.
      • Boretto J.
      • Mattéi M.G.
      • Takahashi M.
      • Jack L.J.
      • Mather I.H.
      • Rouquier S.
      • Pontarotti P.
      ). Recent structural determinations of PRY-SPRY-19q13.4.1, GUSTAVUS, TRIM21, and SOCS box protein 2 (SSB-2) (
      • Grütter C.
      • Briand C.
      • Capitani G.
      • Mittl P.R.
      • Papin S.
      • Tschopp J.
      • Grütter M.G.
      ,
      • Woo J.S.
      • Imm J.H.
      • Min C.K.
      • Kim K.J.
      • Cha S.S.
      • Oh B.H.
      ,
      • James L.C.
      • Keeble A.H.
      • Khan Z.
      • Rhodes D.A.
      • Trowsdale J.
      ,
      • Kuang Z.
      • Yao S.
      • Xu Y.
      • Lewis R.S.
      • Low A.
      • Masters S.L.
      • Willson T.A.
      • Kolesnik T.B.
      • Nicholson S.E.
      • Garrett T.J.
      • Norton R.S.
      ) have shown that this exon encodes the β-sandwich. However, in some cases, additional N-terminal α-helices, encoded by adjacent 5′ exons are an integral part of the entire domain and may play a role in the domain舗s stability. Secondary structure predictions using NPSA and Jpred programs suggest that the equivalent BTN1A1 domain will also contain N-terminal α- helices. Therefore, the term “B30.2” is used throughout this paper to include both the predicted N-terminal α-helices (outside of the historical B30.2 domain) and the β- sandwich (for hBTN1A1, residues 285–476; see Fig. 2 of Ref.
      • Woo J.S.
      • Imm J.H.
      • Min C.K.
      • Kim K.J.
      • Cha S.S.
      • Oh B.H.
      for sequence alignments and further details).
      4The B30.2 domain was first defined as a region of similarity in linear sequence encoded by a single exon in BTN and TRIM protein genes (
      • Vernet C.
      • Boretto J.
      • Mattéi M.G.
      • Takahashi M.
      • Jack L.J.
      • Mather I.H.
      • Rouquier S.
      • Pontarotti P.
      ). Recent structural determinations of PRY-SPRY-19q13.4.1, GUSTAVUS, TRIM21, and SOCS box protein 2 (SSB-2) (
      • Grütter C.
      • Briand C.
      • Capitani G.
      • Mittl P.R.
      • Papin S.
      • Tschopp J.
      • Grütter M.G.
      ,
      • Woo J.S.
      • Imm J.H.
      • Min C.K.
      • Kim K.J.
      • Cha S.S.
      • Oh B.H.
      ,
      • James L.C.
      • Keeble A.H.
      • Khan Z.
      • Rhodes D.A.
      • Trowsdale J.
      ,
      • Kuang Z.
      • Yao S.
      • Xu Y.
      • Lewis R.S.
      • Low A.
      • Masters S.L.
      • Willson T.A.
      • Kolesnik T.B.
      • Nicholson S.E.
      • Garrett T.J.
      • Norton R.S.
      ) have shown that this exon encodes the β-sandwich. However, in some cases, additional N-terminal α-helices, encoded by adjacent 5′ exons are an integral part of the entire domain and may play a role in the domain舗s stability. Secondary structure predictions using NPSA and Jpred programs suggest that the equivalent BTN1A1 domain will also contain N-terminal α- helices. Therefore, the term “B30.2” is used throughout this paper to include both the predicted N-terminal α-helices (outside of the historical B30.2 domain) and the β- sandwich (for hBTN1A1, residues 285–476; see Fig. 2 of Ref.
      • Woo J.S.
      • Imm J.H.
      • Min C.K.
      • Kim K.J.
      • Cha S.S.
      • Oh B.H.
      for sequence alignments and further details).
      is postulated to serve as a protein-binding module, by which proteins interact through the extended surface loops that adjoin individual β-strands (
      • Woo J.S.
      • Suh H.Y.
      • Park S.Y.
      • Oh B.H.
      ).
      One protein that may bind to the cytoplasmic region of BTN proteins (and the B30.2 domain) is the redox enzyme, xanthine oxidoreductase (XOR),
      The enzyme is expressed in most tissues as xanthine dehydrogenase. Conversion to the oxidase form occurs in the mammary gland during or following secretion into milk (see Ref.
      • Ogg S.L.
      • Weldon A.K.
      • Dobbie L.
      • Smith A.J.
      • Mather I.H.
      for discussion). For convenience, the protein is denoted throughout as xanthine oxidoreductase (XOR), regardless of whether it is in the dehydrogenase or the oxidase form. According to international convention, the genes encoding human and bovine BTN1A1 and XOR are denoted BTN1A1 and XDH, respectively, and the mouse genes are denoted Btn1a1 and Xdh, respectively.
      5The enzyme is expressed in most tissues as xanthine dehydrogenase. Conversion to the oxidase form occurs in the mammary gland during or following secretion into milk (see Ref.
      • Ogg S.L.
      • Weldon A.K.
      • Dobbie L.
      • Smith A.J.
      • Mather I.H.
      for discussion). For convenience, the protein is denoted throughout as xanthine oxidoreductase (XOR), regardless of whether it is in the dehydrogenase or the oxidase form. According to international convention, the genes encoding human and bovine BTN1A1 and XOR are denoted BTN1A1 and XDH, respectively, and the mouse genes are denoted Btn1a1 and Xdh, respectively.
      because it was shown to bind to the cytoplasmic domain of mouse BTN1A1 in an in vitro binding assay (
      • Ishii T.
      • Aoki N.
      • Noda A.
      • Adachi T.
      • Nakamura R.
      • Matsuda T.
      ). Furthermore, one XOR-deficient mouse strain (Xdh+/−) (
      • Vorbach C.
      • Scriven A.
      • Capecchi M.R.
      ) displayed a lactation phenotype similar to that of Btn1a1 null mice (
      • Ogg S.L.
      • Weldon A.K.
      • Dobbie L.
      • Smith A.J.
      • Mather I.H.
      ), suggesting that the two proteins may be functionally linked by direct interaction. These conclusions, however, have been challenged, because XOR does not co-localize with BTN1A1 in immunolabeled freeze-fractured replicates of secreted milk lipid droplets (
      • Robenek H.
      • Hofnagel O.
      • Buers I.
      • Lorkowski S.
      • Schnoor M.
      • Robenek M.J.
      • Heid H.
      • Troyer D.
      • Severs N.J.
      ), and a second mouse strain deficient in XOR does not appear to have an altered lactation phenotype (
      • Ohtsubo T.
      • Rovira II
      • Starost M.F.
      • Liu C.
      • Finkel T.
      ).
      In this paper, we devise in vivo and in vitro assays to show that the cytoplasmic domain of BTN1A1 binds to XOR via the B30.2 domain and that BTN1A1 is required for the stable association of XOR with the MFGM in vivo. Furthermore, interaction with XOR appears to be limited to BTN1A1 orthologs. These results are discussed in the context of potential functions of BTN1A1 in the mammary gland and other tissues and the relationship of BTN1A1 to other BTN family members.

      RESULTS

      To directly explore potential interactions between BTN1A1 and XOR, we initially prepared the cytoplasmic domain of mouse BTN1A1 (mBTN1A1cyto) from yeast as a C-terminal fusion protein with GST (GST-mBTN1A1cyto) (supplemental Table 1S). Analysis by SDS-PAGE and two-dimensional gel electrophoresis showed that the purified protein was of the expected size (56,000) (supplemental Fig. 1Sa) and focused as a series of isoelectric variants with pI values between 5.9 and 6.6 (supplemental Fig. 1Sb) (the theoretical pI for GST-mBTN1A1cyto based on amino acid composition is 5.9). The CD spectrum of mBTN1A1cyto, obtained by subtracting the spectrum of GST from that of GST-mBTN1A1cyto, was consistent with a structure rich in β-sheets with no overt structural perturbations (supplemental Fig. 1Sc) (16% α-helix, 35% anti-parallel sheet, 8% parallel sheet, 17% β-turn, and 25% random coil). These values are consistent with a protein in which the B30.2 domain comprises about 75% of the total structure and is predicted to contain one or two α-helices and 13–15 β-strands (
      • Grütter C.
      • Briand C.
      • Capitani G.
      • Mittl P.R.
      • Papin S.
      • Tschopp J.
      • Grütter M.G.
      ,
      • Woo J.S.
      • Imm J.H.
      • Min C.K.
      • Kim K.J.
      • Cha S.S.
      • Oh B.H.
      ,
      • James L.C.
      • Keeble A.H.
      • Khan Z.
      • Rhodes D.A.
      • Trowsdale J.
      ).
      Using the purified and characterized GST-mBTN1A1cyto, we first asked which soluble proteins from lactating mouse mammary gland will bind to mBTN1A1cyto in vitro. Postmicrosomal supernatants obtained from the mammary glands of day 10 lactating mice (Fig. 1, lane 1) were fractionated on either GST or GST-mBTN1A1cyto immobilized on glutathione-agarose, and the bound proteins were eluted with glutathione. The control GST column failed to reproducibly pull down proteins from extracts, and hence only GST was detected after the glutathione wash (Fig. 1, lane 2; protein 6, white asterisk). Specifically bound proteins from the test column with approximate Mr of >200,000, 150,000, 66,000, and 50,000 were among the most prominent detected in the eluates by SDS-PAGE (Fig. 1, lane 3; proteins 1–3 and 5) together with GST-mBTN1A1cyto (Fig. 1, lane 3; protein 4, white asterisk). These proteins were identified as fatty acid synthase, XOR, serum albumin, and eukaryotic elongation factor γ1, respectively, based on peptide analysis by tandem mass spectrometry (Table 1). The 150-kDa protein, identified as XOR, was the most abundant. That GST-mBTN1A1cyto binds to XOR was confirmed by repeating the affinity chromatography with XOR purified from bovine milk (see further data below).
      Figure thumbnail gr1
      FIGURE 1Affinity chromatography of mammary proteins on GST-mBTN1A1cyto. The postmicrosomal supernatant fraction from lactating mouse mammary tissue (3.0 ml; 50 mg of protein) was incubated with either GST or GST-mBTN1A1cyto bound to glutathione-agarose. Interacting proteins were then stripped from the columns using 10 mm glutathione. Samples (120 μg of protein/lane) were separated by electrophoresis in an 8% SDS-polyacrylamide gel. Lane 1, total postmicrosomal supernatant; lane 2, proteins bound to GST; lane 3, proteins bound to GST-mBTN1A1cyto. Representative results from one of three experiments are shown. Proteins 1, 2, 3, and 5 (lane 3) and 6 (lane 2) were identified by electrospray ionization mass spectrometry (). Protein 4 (lane 3) was identified by Western blot. Proteins 6 (lane 2) and 4 (lane 3) (white asterisks) are the baits, GST and GST-mBTN1A1cyto, respectively.
      TABLE 1Identification of proteins binding to mBTN1A1cyto by tandem mass spectrometry
      Protein (Fig. 1)Peptides identifiedProtein identifiedCalculated Mr
      1ALIAEATK, EAVLAAYWR, LTQGEVYKFatty acid synthase272,653
      2FYLTVLQK, YENELSLR, TADELVFFVNGKXOR146,563
      IPAFGSIPTEFR, NADPETTLLAYLR
      3LSQTHPNADFAEITKSerum albumin66,659
      5ALIAAQYSGAQVR, TFLVGERElongation factor γ150,119
      6LLLEYLEEK, IEAIPQIDKGST25,498
      In order to identify the interactive region of mouse BTN1A1 that binds to XOR, we devised two assays, one in which fluorescent fusion proteins of mouse BTN1A1 and mouse XOR were expressed in HEK 293T cells and a second in which mutated GST-mBTN1A1cyto fusion proteins were bound to glutathione-coated beads and used in pull-down assays with bXOR.
      For the in vivo cell expression assay, vectors were constructed encoding mouse BTN1A1 fused at the N terminus (after the signal sequence) to ECFP (pECFP-mBTN1A1) and mXOR fused at the C terminus to EYFP (pmXOR-EYFP) (supplemental Table 1S). When HEK 293T cells were separately transfected with these vectors, ECFP-mBTN1A1 was expressed and sorted to the plasma membrane (Fig. 2A, a) and a variable fraction to an intracellular site. This intracellular site appeared to be associated with or close to the trans-Golgi/trans-Golgi network, because co-expression of mBTN1A1-EYFP and the Golgi marker galactosyltransferase-ECFP showed close association of the two expressed proteins (supplemental Fig. 2SA). In contrast, mXOR-EYFP was expressed in the cytoplasm (Fig. 2A, c). Both expressed mBTN1A1 and mXOR fluorescent fusion proteins were of the expected size, with little evidence of degradation (shown by immunoblots of cell extracts probed with an antibody to GFP; supplemental Fig. 3S). That mXOR-EYFP was primarily expressed in the cytoplasm was confirmed by treating the transfected cells with digitonin to permeabilize the cells and remove cytoplasmic proteins (
      • Lorenz H.
      • Hailey D.W.
      • Lippincott-Schwartz J.
      ). Virtually all of the detectable mXOR-EYFP was washed out of the cells (Fig. 2A, compare c and d). As expected for a membrane protein, the distribution of ECFP-mBTN1A1 was unaffected by digitonin treatment (Fig. 2A, compare a and b).
      Figure thumbnail gr2
      FIGURE 2Example of in vivo binding assay; expression of ECFP-mBTN1A1 and mXOR-EYFP in HEK 293T cells. A, cells were transfected with either pECFP-mBTN1A1 (a and b) or pmXOR-EYFP (c and d) before (-) (a and c), and after (+) (b and d) treatment with digitonin. B, cells were co-transfected with pECFP-mBTN1A1 and pmXOR-EYFP. a, c, and e, before (−) treatment with digitonin; b, d, and f, after (+) treatment with digitonin. Shown is the fluorescence signal for ECFP-mBTN1A1 (a and b) and mXOR-EYFP (c and d). e and f, merged image. C, cells were co-transfected with pECFP-mBTN1A1Δ470–524 and pmXOR-EYFP before (−) (a, c, and e) and after (+) (b, d, and f) treatment with digitonin. Shown is the fluorescence signal for ECFP-mBTN1A1Δ470–524 (a and b) and mXOR-EYFP (c and d). e and f, merged image. Bars, 10 μm.
      To demonstrate binding between the two proteins, HEK 293T cells were co-transfected with both vectors. A fraction of the expressed mXOR-EYFP co-localized with ECFP-mBTN1A1 at the plasma membrane and in the Golgi region, even after treatment with digitonin (Fig. 2B and supplemental Fig. 2SB). That the two proteins were physically bound to each other was confirmed by immunoprecipitating clarified detergent extracts of transfected cells with antibody to mXOR (Fig. 3a). A specific band of protein, which reacted with antibody to mBTN1A1, was detected on blots of immunoprecipitates from doubly transfected cells (Fig. 3a, lane 4) but not from non-transfected control cells (Fig. 3a, lane 1), cells transfected only with mXOR-EYFP (Fig. 3a, lane 2), or cells doubly transfected with mXOR-EYFP and a vector encoding GFP (Fig. 3a, lane 3).
      Figure thumbnail gr3
      FIGURE 3Immunoprecipitation of BTN1A1-XOR complexes in HEK 293T cells and lactating mammary gland. a, HEK 293T cells. Proteins were immunoprecipitated (i.p.) from RIPA buffer extracts with antibody to mXOR, separated by SDS-PAGE (6% gel), and first blotted with antibody to mBTN1A1 (middle blot). The blot was then stripped and reprobed with antibody to mXOR (top blot). Aliquots of the same immunoprecipitates were also separated by SDS-PAGE on a 10% gel to resolve GFP (bottom blot). Immunoprecipitates from non-transfected cells (lane 1), cells transfected with pmXOR-EYFP (lane 2), pmXOR-EYFP and a vector encoding GFP (lane 3), and pmXOR-EYFP and pECFP-mBTN1A1 (lane 4). The RIPA buffer extract used for the immunoprecipitation of mXOR-EYFP and GFP was separated in lane 5 to validate the antibody to GFP (GFP control). b, lactating mammary gland. Proteins were immunoprecipitated from RIPA buffer extracts with antibody to mXOR, separated by SDS-PAGE (6% gel), and first blotted with antibody to mBTN1A1 (bottom blot). The blot was then stripped and reprobed with antibody to mXOR (top blot). Shown are immunoprecipitates from a wild-type mouse (lane 1) and a Btn1a1/ mouse (lane 2). The corresponding RIPA buffer extracts, before and after incubation with the antibody-coated protein A beads, are shown for the wild-type mouse in lanes 3 and 5 and for the Btn1a1/ mouse in lanes 4 and 6, respectively. In both a and b, the blots shown are representative of one of three experiments each.
      Mutated forms of ECFP-mBTN1A1 were then co-expressed with mXOR-EYFP, to determine which region of mBTN1A1cyto is required for localization of both proteins to cellular membranes. The cytoplasmic domain of BTN1A1 comprises three domains: a stem region closest to the membrane anchor, followed by a B30.2 domain and a C-terminal tail region (schematic in Fig. 4). Progressive deletion of the C-terminal region of ECFP-mBTN1A1cyto (supplemental Table 1S) showed that the entire tail domain could be deleted with no effect on binding to mXOR-EYFP (results summarized in Fig. 4). However, the removal of the most C-terminal 10 amino acids of the B30.2 domain resulted in relocation of the ECFP fusion protein to intracellular vesicles and abrogated binding to mXOR-EYFP (Figs. 2C and 4).
      Figure thumbnail gr4
      FIGURE 4Summary of bead binding and cell expression assays for interaction between mBTN1A1cyto and XOR. Regions of mBTN1A1cyto that bound to XOR are indicated by green lines, and regions that did not bind are shown by red lines. Constructs indicated by numbers were inserted into either pECFP-C1 (Clontech) or pESP1 (Stratagene) as follows. For the cell expression assay, the vectors used (supplemental ) were pmXOR-EYFP in combination with pECFP-mBTN1A1 (1), pECFP-mBTN1A1Δ507–524 (2), pECFP-mBTN1A1Δ493–524 (3), pECFP-mBTN1A1Δ480–524 (4), and pECFP-mBTN1A1Δ470–524 (5). For the in vitro bead binding assay, the vectors used were pGST-mBTN1A1cyto (1), pGST-mBTN1A1cytoΔ493–524 (3), pGST-mBTN1A1cytoΔ480–524 (4), pGST-mBTN1A1cytoΔ470–524 (5), pGST-mBTN1A1cytoΔ269–310;480–524 (6), and pGST-mBTN1A1cytoΔ269–285; 480–524 (mB30.2) (7).
      Binding between XOR and the cytoplasmic domain of BTN1A1 was confirmed by glutathione/GST pull-down assays. Fusion proteins of GST joined at the C terminus, either to mBTN1A1cyto or mutated forms of mBTN1A1cyto, were bound to glutathione-coated agarose beads and then incubated with a range of concentrations of bXOR. Binding of bXOR to GST-mBTN1A1cyto was saturable with apparent KD values in the 50 nm range (Fig. 5a and Table 2). Deletion of the tail domain of mBTN1A1cyto had no influence on the binding of bXOR at saturating concentrations (FIGURE 4, FIGURE 5, FIGURE 6a, compare lanes 3 and 4). GST-coated beads or uncoated beads served as controls (Fig. 6a, lanes 1 and 2). However, deletion of 10 amino acids from the C-terminal region of the B30.2 domain or deletion of 25 amino acids from the N-terminal region, abrogated binding (Fig. 6a, lanes 5 and 6). Most significantly, the B30.2 domain alone was sufficient for binding to bXOR (FIGURE 4, FIGURE 5, FIGURE 6a, lane 7).
      Figure thumbnail gr5
      FIGURE 5Summary of bead binding assay for interaction between bXOR and BTN1A1. a, example of binding between GST-mBTN1A1cyto (0.1 nmol) and increasing amounts of bXOR (0–1.0 μm dimer) to determine the KD and stoichiometry at maximal binding; b, example of titration of GST-bB30.2 (0–0.8 nmol) and bXOR (1.0 nmol) to determine stoichiometry of binding. Densitometric data derived from the gels shown in the figure are plotted below each panel. summarizes the binding data for GST-mBTN1A1cyto, GST-bBTN1A1cyto, and GST-bB30.2.
      TABLE 2Summary of binding characteristics for interaction between BTN fusion proteins and bXOR
      ParameterBinding between bXOR and
      GST-mBTN1A1cytoGST-bBTN1A1cytoGST-bB30.2
      Dissociation constants (pH 7.4), KD (nM)
       By bead binding assay47 ± 35 (3)50 ± 27 (3)26 ± 14 (3)
       By SPR113 ± 6.4 (2)112 ± 7.1 (2)101 ± 31 (10)
      Rate constants (pH 7.4)
      ka × 10−4m−1 s−19.2 ± 2.4 (2)7.4 ± 0.30 (2)6.2 ± 1.3 (10)
      kd × 10−3 s−110.4 ± 2.1 (2)8.3 ± 0.15 (2)6.0 ± 1.2 (10)
      Binding stoichiometry (monomers) (BTN1A1:XOR ::1.0:x)
       Binding curve0.72 ± 0.03 (3)0.79 ± 0.04 (3)1.15 ± 0.32 (3)
       Linear titration0.35 ± 0.04 (2)0.94 ± 0.18 (2)1.15 ± 0.13 (2)
      Figure thumbnail gr6
      FIGURE 6In vitro bead binding assay for interaction between bXOR and wild-type or mutant mBTNs or BTNs from different species. Each GST fusion protein (0.5 nmol) was bound to glutathione-coated beads (20-μl packed bed volume) and incubated with 0.5 nmol of bXOR in a final volume of 200 μl of PBS overnight at 4 °C. The beads were washed three times with PBS and heated in SDS-PAGE buffer for separation by SDS-PAGE. a, analysis of binding between bXOR and mutant forms of GST-mBTN1A1cyto. Lane 1, GST; lane 2, no protein; lane 3, GST-mBTN1A1cyto; lane 4, GST-mBTN1A1cytoΔ480–524; lane 5, GST-mBTN1A1 cytoΔ470–524; lane 6, GST-mBTN1A1cytoΔ269–310;480–524; lane 7, GST-mBTN1A1cytoΔ269–285;480–524 (mB30.2). b, specificity of binding between bXOR and different orthologs and paralogs of BTN. Lane 1, GST-bBTN1A1cyto; lane 2, GST-hBTN1A1cyto; lane 3, GST-hBTN1A1-B30.2; lane 4, GST-hBTN2A1cyto; lane 5, GST-hBTN3A1cyto; lane 6, GST-human RoRet(TRIM38)-B30.2; lane 7, no protein. Densitometric analysis of the results from three experiments (mean ± S.D.) are shown below each panel. Band densities are normalized (100%) to GST-mBTN1A1cyto (lane 3) in a and to GST-bBTN1A1cyto (lane 1) in b.
      To investigate binding specificity within and across species, a series of GST fusion proteins were prepared that incorporated, at the C terminus, the entire cytoplasmic domains or the B30.2 domains of bovine and human BTN1A1 or the entire cytoplasmic domains of human BTN2A1 and BTN3A1. In addition, a protein comprising GST fused to the predicted B30.2 domain of human RoRet (TRIM 38) (
      • Ruddy D.A.
      • Kronmal G.S.
      • Lee V.K.
      • Mintier G.A.
      • Quintana L.
      • Domingo Jr., R.
      • Meyer N.C.
      • Irrinki A.
      • McClelland E.E.
      • Fullan A.
      • Mapa F.A.
      • Moore T.
      • Thomas W.
      • Loeb D.B.
      • Harmon C.
      • Tsuchihashi Z.
      • Wolff R.K.
      • Schatzman R.C.
      • Feder J.N.
      ,
      • Meyer M.
      • Gaudieri S.
      • Rhodes D.A.
      • Trowsdale J.
      ) was prepared to test potential binding to a B30.2 domain in a protein outside of the BTN protein family. bXOR bound to the entire cytoplasmic domain or the B30.2 domain of BTN1A1 from mice (see above), cows, or humans (Fig. 6b, lanes 1–3) (data not shown). However, binding was limited to BTN1A1 subfamily members, since no interaction was detected between bXOR and BTN2A1, or BTN3A1 (Fig. 6b, lanes 4 and 5). Furthermore, bXOR did not bind to the B30.2 domain of human RoRet (TRIM 38) (Fig. 6b, lane 6; control in lane 7). These data demonstrate that XOR does not promiscuously bind to the B30.2 domains of even closely related paralogs of BTN1A1.
      We next determined the affinity and kinetics of binding between XOR and BTN1A1 by SPR using GST fusion proteins encoding either bovine or mouse BTN1A1cyto domains as ligands and bXOR as analyte (Table 2 and Fig. 7a, example for GST-bBTN1A1cyto). Dissociation constants (KD values) for GST-BTN1A1cyto from either species, determined at 25 °C, were not significantly different (average of 112 and 113 nm, for cow and mouse, respectively) and consistent with values in the 40–50 nm range for KD values determined by the bead binding assay at 4 °C (Table 2). bXOR bound to GST-bB30.2 with statistically similar affinity (Table 2), confirming that the B30.2 domain is necessary and sufficient for maximal interaction between the two proteins. Binding was pH-dependent (determined either by SPR or the bead binding assay), with no binding detected below pH 6.0 (Fig. 7b), and binding affinities increased 5–7-fold when the salt concentration was lowered from 150 to 20 mm NaCl (Fig. 7c).
      Figure thumbnail gr7
      FIGURE 7Determination of equilibrium and kinetic rate constants for interaction between BTN1A1 proteins and bXOR by SPR. a, representative example of the biosensor analysis of binding of bXOR to GST-bBTN1A1cyto (see for complete summary). bXOR concentrations ranged from 15.6 to 500 nm, as shown in the figure. Data were fitted to the Langmuir model for 1:1 binding (black lines). b, effect of pH on binding of bXOR to GST-bB30.2. Solid line, SPR analysis (KA μm−1). Dotted lines, bead binding assay, using the following buffers: KH2PO4-KOH, pH 4.5–5.5 (red); MES, pH 5.5–7.0 (purple); HEPES, pH 7.0–8.0 (green); and Tricine, pH 8.0–9.0 (blue). c, effect of NaCl on the binding of bXOR to GST-bB30.2 (KA μm−1).
      In order to determine the stoichiometry of binding between BTN1A1 and XOR, we titrated GST fusion proteins bound to glutathione-coated beads with bXOR, either keeping the amount of GST fusion protein constant and varying the amount of bXOR (binding curve) or varying the amount of GST fusion protein on the beads and adding saturating amounts of bXOR (linear titration) and then assaying for the amount of bXOR bound to the beads by quantitative densitometry (Fig. 5, a and b, and Table 2). Either approach gave BTN1A1/bXOR ratios consistent with equimolar binding (1.0:1.0), with the exception of the linear titration of GST-mBTN1A1cyto, which for unexplained reasons gave a ratio of 1.0:0.36. Binding was equimolar for both full-length mouse and bovine BTN1A1cyto or just the bB30.2 domain. Since bXOR is a homodimer (
      • Enroth C.
      • Eger B.T.
      • Okamoto K.
      • Nishino T.
      • Nishino T.
      • Pai E.F.
      ), these data indicate that one XOR dimer binds to either two BTN1A1 monomers or one BTN1A1 dimer.
      The oligomeric state of BTN1A1 and further estimates for stoichiometric ratios were investigated by gel filtration on Superdex 200 (FPLC) to determine the apparent Mr values of the purified proteins, separately, and together as BTN1A1-XOR complexes (Fig. 8 and Table 3). Both GST-mBTN1A1cyto and GST-bBTN1A1cyto eluted as major peaks of protein with apparent Mr values in the range expected for tetramers (∼190,000 and 215,000, respectively) (Table 3; see Fig. 8, a and b, dotted blue lines, for examples of GST-bBTN1A1cyto). Since GST is a dimer (
      • Lim K.
      • Ho J.X.
      • Keeling K.
      • Gilliland G.L.
      • Ji X.
      • Rüker F.
      • Carter D.C.
      ), we removed GST from GST-bBTN1A1cyto by digestion with thrombin to determine if the ability to oligomerize is an intrinsic property of BTN1A1cyto. The entire cytoplasmic domain of bBTN1A1, eluted with an apparent Mr of 48,000, ∼80% of the mass expected for a dimer (61,020) (Fig. 8b, dotted red line, and Table 3). However, the purified bB30.2 domain eluted with an estimated Mr of 19,500, close to that expected for a monomer (23,437), with a lower amount of potential dimer partially separating as a shoulder on the major peak of protein (Fig. 8b, solid red line, and Table 3). These data are consistent with oligomerization of the fusion proteins to tetramers through separate dimeric interactions between the GST domains and dimeric interactions between the cytoplasmic domains of BTN1A1. Furthermore, interactions between the BTN molecules appear to be predominantly due to interactions within either the stem or tail regions, since the B30.2 domain was largely monomeric.
      Figure thumbnail gr8
      FIGURE 8Gel filtration of protein complexes. Samples were separated by FPLC on a 1 × 30-cm column of Superdex 200 in TBS, pH 7.4, and protein was detected at A280 nm (light path, 0.5 cm). a, GST-bBTN1A1cyto (10 nmol) (dotted blue line) and GSTbBTN1A1-bXOR complex (10 nmol) (black line). The gel inset shows analysis of peak fractions by SDS-PAGE; b, GST-bBTN1A1cyto (8 nmol) (dotted blue line), bBTN1A1cyto (8 nmol) (dotted red line), and bB30.2 (8 nmol) (solid red line). c, GSTbB30.2-bXOR complex; gel inset shows analysis of peak fraction by SDS-PAGE (lane 2) in comparison with protein standards (from top to bottom, 250, 150, 100, 75, 50, 37, 25, and 20 kDa; lane 1).
      TABLE 3Protein Mr values estimated by gel filtration on Superdex 200
      ProteinMr × 10−3
      MouseCow
      GST-BTN1A1cyto; Mr expected for monomer189 ± 19 (5); 55.92216 ± 4.2 (3); 56.51
      BTN1A1cyto; Mr expected for monomerND48 ± 3.1 (3); 30.51
      B30.2; Mr expected for monomerND[30.8], 19.5 (1); 23.44
      GST-BTN1A1cyto-bXOR complex[764 ± 33] (5)[807 ± 10] (3)
      496 ± 29528 ± 8
      B30.2-bXOR complexND344 ± 13 (3)
      Complexes of the GST fusion proteins and bXOR, which had been prepared by binding and elution from glutathione-coated beads, eluted as broad peaks of protein with apparent Mr of ∼500,000 with significant peak shoulders of ∼800,000 (Table 3; Fig. 8a, solid black line, example for GST-bBTN1A1). These data are consistent with stoichiometries of one tetrameric GST-fusion protein (Mr ∼225,000) binding to either one or two dimers of XOR (expected Mr values for the complex of 520,000 and 815,000, respectively). To eliminate the confounding effects of dimeric GST, complexes of GST-bB30.2 and bXOR were prepared on glutathione-coated beads, and the GST was removed by digestion with thrombin. Such bB30.2-bXOR complexes eluted with an apparent Mr of 344 ± 13 (Fig. 8c and Table 3) (i.e. with a predicted monomeric bB30.2/XOR ratio of 1:0.85). Taken together, these data confirm that the cytoplasmic domain of BTN1A1 binds to bXOR in equimolar amounts via the B30.2 domain.
      Finally, to assess the potential function of BTN1A1 and XOR in lactation, we first showed that the two proteins bind to each other in vivo. Detergent extracts of membrane fractions, prepared from the lactating mammary tissue of either wild type (Btn1a1+/+) or “knock-out” (Btn1a1/) mice, were immunoprecipitated with antibody to mXOR. A band of protein that reacted with antibody to mBTN1A1 was detected when the immunoprecipitates from wild-type mice were analyzed by Western blot using antibody to mBTN1A1 (Fig. 3b, lane 1), whereas there was no reaction with immunoprecipitates from Btn1a1/ mice (Fig. 3b, lane 2). ∼37.5 ± 6.2% of BTN1A1 (mean and S.D. of three determinations) was bound to XOR, as determined by densitometric analysis of the detergent extracts before and after incubation with the antibody-coated protein A beads (Fig. 3b, lanes 3 and 5). Interestingly, XOR was recovered in the immunoprecipitates from knock-out mice, indicating that XOR binds to membranes in the absence of BTN1A1 (Fig. 3b, lane 2).
      To determine whether the binding of XOR to BTN1A1 has relevance to the potential function of these proteins in milk lipid secretion, we compared the association of XOR with lipid droplets in the presence and absence of BTN1A1 using wild-type, heterozygous (Btn1a1+/−), or Btn1a1/ mice. No significant differences in the amount of XOR relative to total protein (determined by immunoblot) or activity (determined as xanthine oxidase activity) were detected in lipid droplets from the milks of wild-type, Btn1a1+/−, or Btn1a1/ mice (Fig. 9). However, although the levels of XOR were comparable between wild-type and Btn1a1/ mice, there was a highly significant difference in the amount of XOR that remained bound to the MFGM compared with the amount of XOR that was released in a soluble form when membrane was stripped from the core lipid by freeze-thawing the cream samples. Approximately four times as much XOR remained bound to membrane compared with the soluble fraction in wild-type mice, whereas a majority of the XOR in both Btn1a1+/− and Btn1a1/ mice was released into the soluble fraction (Fig. 9, b and c). Furthermore, a fraction of the membrane-bound XOR in wild-type mice separated as aggregates when samples were separated by SDS-PAGE, compared with fractions from Btn1a1/ mice (bracket in Fig. 9a, i). At least some of these aggregates may consist of complexes of XOR and BTN1A1, because both proteins were detected in the same protein bands by stripping the immunoblots and reprobing them with an antibody to BTN1A1; filled circle in Fig. 9a, ii). Thus, XOR is incorporated into secreted lipid droplets in the absence of BTN1A1 but is more loosely bound to the MFGM. These data are consistent with the possibility that association of XOR with the MFGM is stabilized by binding to the cytoplasmic domain of BTN1A1, but XOR is initially incorporated into lipid droplets by another mechanism.
      Figure thumbnail gr9
      FIGURE 9Distribution and amount of XOR in milk lipid droplets and MFGM fractions from Btn1a1+/+, Btn1a1+/−, and Btn1a1−/− mice at peak lactation. Milk lipid fractions were separated into membrane-bound and soluble fractions as described under “Experimental Procedures” and analyzed by Western blot (a and b) and an enzyme assay (c). a (i), membrane-bound (m) and soluble (s) XOR detected by Western blot. a (ii), the same immunoblots from a (i), stripped and reprobed with an antibody to mouse BTN1A1. Aggregates of XOR and BTN1A1 are indicated by brackets in i and ii, respectively, and potential complexes of XOR and BTN1A1 are shown by filled circles. b, left, comparative amounts of total XOR determined by densitometry of stained immunoblots; right, ratio of membrane-bound to soluble XOR. c, left, comparative amounts of total XOR activity; right, ratio of membrane-bound to soluble XOR activity. For each genotype, milk lipid samples from three mice on day 10 of lactation were analyzed in duplicate. Data within each panel with different letters are significantly different from each other (p < 0.05) using Student-Newman-Keul舗s multiple comparison test.

      DISCUSSION

      The data described firmly establish that the cytoplasmic domain of BTN1A1 binds to XOR in vivo and in vitro with relatively high affinity and in a salt- and pH- dependent manner via the B30.2 domain. Binding is stoichiometric with predicted ratios of one XOR homodimer bound to two BTN monomers or one BTN1A1 dimer. Furthermore, binding is independent of species; bovine or mouse XOR binds to the B30.2 domain of mouse, cow, or human BTN1A1. No binding was detected between XOR and even the closely related paralogs, BTN2A1 and BTN3A1, or to the B30.2 domain of the otherwise unrelated RoRet (TRIM 38) protein. Analysis of Btn1a1/ mice showed that expression of BTN1A1 is required for the stable association of XOR with the MFGM in vivo. These data provide novel insights into the potential roles of BTN1A1 and XOR in milk lipid secretion, the relationship between BTN1A1 and other family members, and the possible functions of BTN1A1 in other cellular contexts.
      Based on available structural data, there is an emerging consensus that the B30.2 domain serves as a universal protein binding module (
      • Grütter C.
      • Briand C.
      • Capitani G.
      • Mittl P.R.
      • Papin S.
      • Tschopp J.
      • Grütter M.G.
      ,
      • Woo J.S.
      • Imm J.H.
      • Min C.K.
      • Kim K.J.
      • Cha S.S.
      • Oh B.H.
      ,
      • Woo J.S.
      • Suh H.Y.
      • Park S.Y.
      • Oh B.H.
      ,
      • James L.C.
      • Keeble A.H.
      • Khan Z.
      • Rhodes D.A.
      • Trowsdale J.
      ,
      • Kuang Z.
      • Yao S.
      • Xu Y.
      • Lewis R.S.
      • Low A.
      • Masters S.L.
      • Willson T.A.
      • Kolesnik T.B.
      • Nicholson S.E.
      • Garrett T.J.
      • Norton R.S.
      ,
      • Masters S.L.
      • Yao S.
      • Willson T.A.
      • Zhang J.G.
      • Palmer K.R.
      • Smith B.J.
      • Babon J.J.
      • Nicola N.A.
      • Norton R.S.
      • Nicholson S.E.
      ). The B30.2 domain is widely distributed throughout the proteome (
      • Rhodes D.A.
      • de Bono B.
      • Trowsdale J.
      ,
      • Henry J.
      • Mather I.H.
      • McDermott M.F.
      • Pontarotti P.
      ,
      • Laing K.J.
      • Purcell M.K.
      • Winton J.R.
      • Hansen J.D.
      ), occurring within and outside of the BTN protein family and the immune system. The most conserved region of the domain comprises a core β-sandwich, with variable loops linking adjoining β-strands. In all B30.2 domain structures that have been characterized with bound ligand (
      • Woo J.S.
      • Imm J.H.
      • Min C.K.
      • Kim K.J.
      • Cha S.S.
      • Oh B.H.
      ,
      • Woo J.S.
      • Suh H.Y.
      • Park S.Y.
      • Oh B.H.
      ,
      • James L.C.
      • Keeble A.H.
      • Khan Z.
      • Rhodes D.A.
      • Trowsdale J.
      ), the interacting partner binds, via hydrogen bonding and hydrophobic interactions, to one or more discrete pockets formed from the variable loops. Comparison of the linear sequences of these B30.2 domains with those of BTN1A1 and other family members (BTN2A1, BTN2A2, BTN2A3, BTN3A1, BTN3A2, and BTN3A3) reveals a similar predicted pattern of conserved β-strands and variable loops (
      • Woo J.S.
      • Imm J.H.
      • Min C.K.
      • Kim K.J.
      • Cha S.S.
      • Oh B.H.
      ,
      • James L.C.
      • Keeble A.H.
      • Khan Z.
      • Rhodes D.A.
      • Trowsdale J.
      ). XOR is therefore likely to interact with BTN1A1 through analogous binding motifs, which are not conserved in BTN2A1 or BTN3A1 (Fig. 6b). Similar binding characteristics are also to be expected because of similarities in the dissociation constants (KD values) for the binding of ligand to BTN1A1 (27 nm in 20 mm NaCl), GUSTAVUS (40 nm in 150 mm NaCl) (
      • Woo J.S.
      • Imm J.H.
      • Min C.K.
      • Kim K.J.
      • Cha S.S.
      • Oh B.H.
      ), and TRIM 21 (37 nm in 50 mm NaCl) (
      • James L.C.
      • Keeble A.H.
      • Khan Z.
      • Rhodes D.A.
      • Trowsdale J.
      ) and the lower KD values (higher affinities) determined at lower salt concentrations (Fig. 7c) (
      • James L.C.
      • Keeble A.H.
      • Khan Z.
      • Rhodes D.A.
      • Trowsdale J.
      ). However, because of the presumed discontinuous nature of binding motifs in the linear sequence, it is not possible to predict which residues in BTN1A1 function in binding. Resolution of these structural issues will require analysis by x-ray crystallography.
      In vitro bead binding assays showed that bXOR binds to the B30.2 domain of BTN1A1 in a monomeric ratio of 1:1 (Fig. 5 and Table 2), which is in agreement with a global best fit of the SPR data using the 1:1 Langmuir model for binding (Fig. 7a, black lines). These data are also consistent with ligand binding ratios for other B30.2 domain-containing proteins, including GUSTAVUS and TRIM21 (
      • Woo J.S.
      • Imm J.H.
      • Min C.K.
      • Kim K.J.
      • Cha S.S.
      • Oh B.H.
      ,
      • James L.C.
      • Keeble A.H.
      • Khan Z.
      • Rhodes D.A.
      • Trowsdale J.
      ). Since bXOR is a homodimer (
      • Enroth C.
      • Eger B.T.
      • Okamoto K.
      • Nishino T.
      • Nishino T.
      • Pai E.F.
      ), a maximum of two B30.2 domains are predicted to bind to one native XOR molecule. In vitro analysis by FPLC showed that bBTN1A1cyto can form dimers. Dimerization is most likely through the stem region, which is predicted to have α-helical character, since the B30.2 domain was largely monomeric (Fig. 8b), and the cytoplasmic tail domain is probably disordered because of intermittent P residues (GlobPlotTM prediction). Therefore, XOR has the potential to bind either one or two BTN1A1 monomers or one dimer in a pH-dependent manner.
      These binding data are relevant to several potential functions of BTN1A1 in vivo. In the context of milk lipid secretion, fat droplets are coated with apical membrane as they are expelled from mammary cells, and the resulting MFGM in many species contains large amounts of BTN1A1 and XOR (
      • Mather I.H.
      • Keenan T.W.
      ,
      • Heid H.W.
      • Winter S.
      • Bruder G.
      • Keenan T.W.
      • Jarasch E.D.
      ,
      • Mather I.H.
      ). The observation that XOR only binds to BTN1A1, the BTN family member that is highly expressed in the lactating mammary gland, and not to other BTN paralogs provides further evidence that these two proteins function together in lactation. At least two models have been proposed to explain how BTN1A1 and XOR may function in lipid secretion (
      • Mather I.H.
      • Keenan T.W.
      ,
      • Robenek H.
      • Hofnagel O.
      • Buers I.
      • Lorkowski S.
      • Schnoor M.
      • Robenek M.J.
      • Heid H.
      • Troyer D.
      • Severs N.J.
      ,
      • McManaman J.L.
      • Russell T.D.
      • Schaack J.
      • Orlicky D.J.
      • Robenek H.
      ).
      In one model, BTN1A1 as a transmembrane protein binds to XOR, which serves as a linker protein to other proteins on the lipid droplet surface, including adipophilin (
      • Mather I.H.
      • Keenan T.W.
      ). Concerted interactions between such BTN-XOR complexes and homophilic adhesive interactions between BTN1A1 molecules are proposed to proceed as a wave around the droplet and lead to the uniform coating of the droplet with membrane and to the eventual expulsion of the lipid droplet from the cell. At some point during or after secretion, associations between BTN1A1 and XOR culminate in formation of a characteristic protein coat complex that can be identified by freeze-etch electron microscopy (
      • Buchheim W.
      ,
      • Wooding F.B.P.
      ). Knock-out of BTN1A1 expression in Btn1a1/ mice (
      • Ogg S.L.
      • Weldon A.K.
      • Dobbie L.
      • Smith A.J.
      • Mather I.H.
      ) or reduction of the amount of XOR in one strain of Xdh+/− mice (
      • Vorbach C.
      • Scriven A.
      • Capecchi M.R.
      ) eliminates or reduces the possibility of interactions between BTN1A1 and XOR, leading to the secretion of abnormally large and unstable lipid droplets. Thus, BTN1A1 is seen to function as a transmembrane scaffold, which binds to XOR and other proteins on the droplet surface to ensure the coordinated and regulated secretion of lipid droplets into milk.
      The binding data from this current study confirm that BTN1A1, through interactions with the B30.2 domain, binds with high affinity to XOR and that BTN1A1 molecules can bind to each other. However, the observation that XOR is secreted with lipid droplets in the absence of BTN1A1 (Fig. 9) is not compatible with the proposal that BTN1A1 directly recruits XOR into the lipid droplet (
      • Mather I.H.
      • Keenan T.W.
      ) and suggests that XOR is initially recruited into the MFGM by other means (e.g. by being carried to the apical plasma membrane on the surface of intracellular lipid droplets independently of BTN1A1 or by binding to other proteins as well as BTN1A1) (see Refs.
      • McManaman J.L.
      • Russell T.D.
      • Schaack J.
      • Orlicky D.J.
      • Robenek H.
      and
      • McManaman J.L.
      • Palmer C.A.
      • Wright R.M.
      • Neville M.C.
      for discussion). Interestingly, XOR was recovered in crude membrane fractions (Fig. 3b, lane 4) and the MFGM (Fig. 9a, i) in the absence of BTN1A1, indicating that XOR can bind to other membrane components. However, such associations may be artifactually induced in vitro, because XOR has the potential to bind to glycoaminoglycans on exoplasmic membrane surfaces (
      • Adachi T.
      • Fukushima T.
      • Usami Y.
      • Hirano K.
      ) when it is released into the soluble phase from total cell lysates or homogenized milk lipid droplets. Whatever the case, at some point during the secretion process, association of XOR with the MFGM becomes dependent on the presence of BTN1A1, because its elimination in null mice leads to the release of a substantial amount of soluble XOR from isolated milk lipid globules (Fig. 9). A full description of how the MFGM is assembled will require the application of live cell imaging techniques, more refined analysis of the distribution of XOR and BTN1A1 in secretory epithelial cells, and identification of additional protein binding partners and regulatory molecules. Such additional proteins may include fatty acid synthetase, the γ-subunit of elongation factor 1 (Table 1), and other GTP-binding proteins (
      • Keon B.H.
      • Ankrapp D.P.
      • Keenan T.W.
      ).
      In a second model, based on freeze-fracture labeling techniques, Robenek et al. (
      • Robenek H.
      • Hofnagel O.
      • Buers I.
      • Lorkowski S.
      • Schnoor M.
      • Robenek M.J.
      • Heid H.
      • Troyer D.
      • Severs N.J.
      ) proposed that secretion of lipid droplets is solely mediated by adhesive interactions between BTN1A1 molecules in the apical plasma membrane and BTN1A1 molecules on the lipid droplet surface and that XOR plays no role at any stage, because it does not colocalize with BTN1A1 in the freeze-fractured faces of secreted lipid droplets. This model is not consistent with the data in this paper showing that BTN1A1 binds tightly to XOR and that the association of XOR with the MFGM is dependent upon the presence of BTN1A1 (Fig. 9). Furthermore, it is inconsistent with the data of McManaman et al. (
      • McManaman J.L.
      • Palmer C.A.
      • Wright R.M.
      • Neville M.C.
      ) showing that at least a fraction of BTN1A1 may bind to XOR by disulfide bonds in isolated MFGM. Also, the model assumes that BTN1A1, which is an integral membrane protein, binds with unconventional topology to the phospholipid monolayer surrounding cytoplasmic lipid droplets.
      Historically, BTN1A1 has been considered to be a mammary-specific protein (
      • Franke W.W.
      • Heid H.W.
      • Grund C.
      • Winter S.
      • Freudenstein C.
      • Schmid E.
      • Jarasch E.D.
      • Keenan T.W.
      ), and its functions have been thought to be limited to potential roles in milk lipid secretion, a process unique to the mammary gland (
      • Mather I.H.
      • Keenan T.W.
      ). However, extensive tissue surveys by more sensitive histochemical (BTN1A1 on the Human Protein Atlas site on the World Wide Web) and reverse transcription-PCR techniques
      I. A. Smith, B. R. Knezevic, J. Ammann, D. A. Rhodes, D. Aw, D. B. Palmer, I. H. Mather, and J. Trowsdale, unpublished observations.
      indicate that BTN1A1 is expressed in other cells and tissues, including spleen, thymus, esophagus, and lymphoid cells in the tonsil. Since none of these other tissues secrete membrane-coated lipid droplets, BTN1A1 must function in other capacities. Two possibilities deserve special consideration. First, BTN1A1 and its binding partner, XOR, possess all of the hallmarks of a novel signaling system; BTN1A1 is an integral protein with IgG folds in the exoplasmic domain, which could bind ligand, and a cytoplasmic B30.2 domain, which binds XOR in a pH-dependent manner (this study). In the appropriate physiological contexts, XOR, either as a dehydrogenase or an oxidase may generate short lived reactive species, including H2O2, superoxide radical, and NO, which may act on downstream targets and regulate gene expression (
      • Pahl H.L.
      • Baeuerle P.A.
      ). Second, BTN1A1 and XOR (
      • Martin H.M.
      • Hancock J.T.
      • Salisbury V.
      • Harrison R.
      ,
      • Vorbach C.
      • Harrison R.
      • Capecchi M.R.
      ) may function as components of the innate immune system by generating antibacterial reactive oxygen and nitrogen species following engagement of BTN1A1 with immune receptors or bacterial surfaces. In this context, milk contains large numbers of exosomes, small immunoregulatory 30–100-nm vesicles that contain significant amounts of both BTN1A1 and XOR (
      • Admyre C.
      • Johansson S.M.
      • Qazi K.R.
      • Filén J.J.
      • Lahesmaa R.
      • Norman M.
      • Neve E.P.
      • Scheynius A.
      • Gabrielsson S.
      ), which may serve to protect the lactating mammary gland from bacterial pathogens.
      The SPRY and B30.2 domains have been identified in at least 86 human genes (
      • Rhodes D.A.
      • de Bono B.
      • Trowsdale J.
      ). In other organisms, well over 2,000 sequences encoding B30.2, SPRY, and PRY domains have been cataloged, including several hundred NOD-like immune receptors in teleost fish (
      • Laing K.J.
      • Purcell M.K.
      • Winton J.R.
      • Hansen J.D.
      ). Although XOR only bound to the B30.2 domain of BTN1A1 in our limited survey of potential binding partners, a more extensive screen is warranted, to determine whether XOR binds to the B30.2 domain in other proteins within and outside of the immune system.

      Acknowledgments

      We thank Drs. Jennifer Lippincott-Schwartz and Dale Hailey (National Institutes of Health, Bethesda, MD) for modified Clontech vectors encoding monomerized forms of ECFP and EYFP and galactosyltransferase-ECFP and Dr. David Rhodes (University of Cambridge, UK) for cDNAs encoding hBTN2A1, hBTN3A1, and human RoRet. Dr. Yinghua Zhang (University of Maryland Medical School, Baltimore, MD) conducted the SPR spectroscopy on a fee for service basis, and Drs. Philip Bryan (Center for Advanced Research in Biotechnology, Gaithersburg, MD) and Victor Munoz (Department of Chemistry and Biochemistry, University of Maryland, College Park, MD) provided help and advice with the CD spectroscopy. Liane Langbehn provided animal care and helped with the preparation of rabbit antibody to bXOR.

      REFERENCES

        • Ogg S.L.
        • Weldon A.K.
        • Dobbie L.
        • Smith A.J.
        • Mather I.H.
        Proc. Natl. Acad. Sci. U.S.A. 2004; 101: 10084-10089
        • Mather I.H.
        • Keenan T.W.
        J. Mammary Gland Biol. Neoplasia. 1998; 3: 259-273
        • Valentonyte R.
        • Hampe J.
        • Huse K.
        • Rosenstiel P.
        • Albrecht M.
        • Stenzel A.
        • Nagy M.
        • Gaede K.I.
        • Franke A.
        • Haesler R.
        • Koch A.
        • Lengauer T.
        • Seegert D.
        • Reiling N.
        • Ehlers S.
        • Schwinger E.
        • Platzer M.
        • Krawczak M.
        • Müller-Quernheim J.
        • Schürmann M.
        • Schreiber S.
        Nat. Genet. 2005; 37: 357-364
        • Nguyen T.
        • Liu X.K.
        • Zhang Y.
        • Dong C.
        J. Immunol. 2006; 176: 7354-7360
        • Arnett H.A.
        • Escobar S.S.
        • Gonzalez-Suarez E.
        • Budelsky A.L.
        • Steffen L.A.
        • Boiani N.
        • Zhang M.
        • Siu G.
        • Brewer A.W.
        • Viney J.L.
        J. Immunol. 2007; 178: 1523-1533
        • Boyden L.M.
        • Lewis J.M.
        • Barbee S.D.
        • Bas A.
        • Girardi M.
        • Hayday A.C.
        • Tigelaar R.E.
        • Lifton R.P.
        Nat. Genet. 2008; 40: 656-662
        • Stefferl A.
        • Schubart A.
        • Storch M.
        • Amini A.
        • Mather I.
        • Lassmann H.
        • Linington C.
        J. Immunol. 2000; 165: 2859-2865
        • Guggenmos J.
        • Schubart A.S.
        • Ogg S.
        • Andersson M.
        • Olsson T.
        • Mather I.H.
        • Linington C.
        J. Immunol. 2004; 172: 661-668
        • Mañá P.
        • Goodyear M.
        • Bernard C.
        • Tomioka R.
        • Freire-Garabal M.
        • Liñares D.
        Int. Immunol. 2004; 16: 489-499
        • Gardinier M.V.
        • Amiguet P.
        • Linington C.
        • Matthieu J.M.
        J. Neurosci. Res. 1992; 33: 177-187
        • Linsley P.S.
        • Peach R.
        • Gladstone P.
        • Bajorath J.
        Protein Sci. 1994; 3: 1341-1343
        • Vernet C.
        • Boretto J.
        • Mattéi M.G.
        • Takahashi M.
        • Jack L.J.
        • Mather I.H.
        • Rouquier S.
        • Pontarotti P.
        J. Mol. Evol. 1993; 37: 600-612
        • Ponting C.
        • Schultz J.
        • Bork P.
        Trends Biochem. Sci. 1997; 22: 193-194
        • Jack L.J.
        • Mather I.H.
        J. Biol. Chem. 1990; 265: 14481-14486
        • Franke W.W.
        • Heid H.W.
        • Grund C.
        • Winter S.
        • Freudenstein C.
        • Schmid E.
        • Jarasch E.D.
        • Keenan T.W.
        J. Cell Biol. 1981; 89: 485-494
        • Harpaz Y.
        • Chothia C.
        J. Mol. Biol. 1994; 238: 528-539
        • Breithaupt C.
        • Schubart A.
        • Zander H.
        • Skerra A.
        • Huber R.
        • Linington C.
        • Jacob U.
        Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 9446-9451
        • Malcherek G.
        • Mayr L.
        • Roda-Navarro P.
        • Rhodes D.
        • Miller N.
        • Trowsdale J.
        J. Immunol. 2007; 179: 3804-3811
        • Compte E.
        • Pontarotti P.
        • Collette Y.
        • Lopez M.
        • Olive D.
        Eur. J. Immunol. 2004; 34: 2089-2099
        • Grütter C.
        • Briand C.
        • Capitani G.
        • Mittl P.R.
        • Papin S.
        • Tschopp J.
        • Grütter M.G.
        FEBS Lett. 2006; 580: 99-106
        • Woo J.S.
        • Imm J.H.
        • Min C.K.
        • Kim K.J.
        • Cha S.S.
        • Oh B.H.
        EMBO J. 2006; 25: 1353-1363
        • Woo J.S.
        • Suh H.Y.
        • Park S.Y.
        • Oh B.H.
        Mol. Cell. 2006; 24: 967-976
        • James L.C.
        • Keeble A.H.
        • Khan Z.
        • Rhodes D.A.
        • Trowsdale J.
        Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 6200-6205
        • Kuang Z.
        • Yao S.
        • Xu Y.
        • Lewis R.S.
        • Low A.
        • Masters S.L.
        • Willson T.A.
        • Kolesnik T.B.
        • Nicholson S.E.
        • Garrett T.J.
        • Norton R.S.
        J. Mol. Biol. 2009; 386: 662-674
        • Rhodes D.A.
        • de Bono B.
        • Trowsdale J.
        Immunology. 2005; 116: 411-417
        • Ishii T.
        • Aoki N.
        • Noda A.
        • Adachi T.
        • Nakamura R.
        • Matsuda T.
        Biochim. Biophys. Acta. 1995; 1245: 285-292
        • Vorbach C.
        • Scriven A.
        • Capecchi M.R.
        Genes Dev. 2002; 16: 3223-3235
        • Robenek H.
        • Hofnagel O.
        • Buers I.
        • Lorkowski S.
        • Schnoor M.
        • Robenek M.J.
        • Heid H.
        • Troyer D.
        • Severs N.J.
        Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 10385-10390
        • Ohtsubo T.
        • Rovira II
        • Starost M.F.
        • Liu C.
        • Finkel T.
        Circ. Res. 2004; 95: 1118-1124
        • Wood V.
        • Gwilliam R.
        • Rajandream M.A.
        • Lyne M.
        • Lyne R.
        • Stewart A.
        • Sgouros J.
        • Peat N.
        • Hayles J.
        • Baker S.
        • Basham D.
        • Bowman S.
        • Brooks K.
        • Brown D.
        • Brown S.
        • Chillingworth T.
        • Churcher C.
        • Collins M.
        • Connor R.
        • Cronin A.
        • Davis P.
        • Feltwell T.
        • Fraser A.
        • Gentles S.
        • Goble A.
        • Hamlin N.
        • Harris D.
        • Hidalgo J.
        • Hodgson G.
        • Holroyd S.
        • Hornsby T.
        • Howarth S.
        • Huckle E.J.
        • Hunt S.
        • Jagels K.
        • James K.
        • Jones L.
        • Jones M.
        • Leather S.
        • McDonald S.
        • McLean J.
        • Mooney P.
        • Moule S.
        • Mungall K.
        • Murphy L.
        • Niblett D.
        • Odell C.
        • Oliver K.
        • o舗Neil S.
        • Pearson D.
        • Quail M.A.
        • Rabbinowitsch E.
        • Rutherford K.
        • Rutter S.
        • Saunders D.
        • Seeger K.
        • Sharp S.
        • Skelton J.
        • Simmonds M.
        • Squares R.
        • Squares S.
        • Stevens K.
        • Taylor K.
        • Taylor R.G.
        • Tivey A.
        • Walsh S.
        • Warren T.
        • Whitehead S.
        • Woodward J.
        • Volckaert G.
        • Aert R.
        • Robben J.
        • Grymonprez B.
        • Weltjens I.
        • Vanstreels E.
        • Rieger M.
        • Schäfer M.
        • Müller-Auer S.
        • Gabel C.
        • Fuchs M.
        • Düsterhöft A.
        • Fritzc C.
        • Holzer E.
        • Moestl D.
        • Hilbert H.
        • Borzym K.
        • Langer I.
        • Beck A.
        • Lehrach H.
        • Reinhardt R.
        • Pohl T.M.
        • Eger P.
        • Zimmermann W.
        • Wedler H.
        • Wambutt R.
        • Purnelle B.
        • Goffeau A.
        • Cadieu E.
        • Dreano S.
        • Gloux S.
        • Lelaure V.
        • Mottier S.
        • Galibert F.
        • Aves S.J.
        • Xiang Z.
        • Hunt C.
        • Moore K.
        • Hurst S.M.
        • Lucas M.
        • Rochet M.
        • Gaillardin C.
        • Tallada V.A.
        • Garzon A.
        • Thode G.
        • Daga R.R.
        • Cruzado L.
        • Jimenez J.
        • Sanchez M.
        • del Rey F.
        • Benito J.
        • Dominguez A.
        • Revuelta J.L.
        • Moreno S.
        • Armstrong J.
        • Forsburg S.L.
        • Cerutti L.
        • Lowe T.
        • McCombie W.R.
        • Paulsen I.
        • Potashkin J.
        • Shpakovski G.V.
        • Ussery D.
        • Barrell B.G.
        • Nurse P.
        Nature. 2002; 415: 871-880
        • Laemmli U.K.
        Nature. 1970; 227: 680-685
        • Neira L.M.
        • Mather I.H.
        Protoplasma. 1990; 159: 168-178
        • Towbin H.
        • Staehelin T.
        • Gordon J.
        Proc. Natl. Acad. Sci. U.S.A. 1979; 76: 4350-4354
        • Banghart L.R.
        • Chamberlain C.W.
        • Velarde J.
        • Korobko I.V.
        • Ogg S.L.
        • Jack L.J.
        • Vakharia V.N.
        • Mather I.H.
        J. Biol. Chem. 1998; 273: 4171-4179
        • Sullivan C.H.
        • Mather I.H.
        • Greenwalt D.E.
        • Madara P.J.
        Mol. Cell Biochem. 1982; 44: 13-22
        • Kreis T.E.
        EMBO J. 1986; 5: 931-941
        • Böhm G.
        • Muhr R.
        • Jaenicke R.
        Protein Eng. 1992; 5: 191-195
        • Gill S.C.
        • von Hippel P.H.
        Anal. Biochem. 1989; 182: 319-326
        • Shevchenko A.
        • Wilm M.
        • Vorm O.
        • Mann M.
        Anal. Chem. 1996; 68: 850-858
        • Smith P.K.
        • Krohn R.I.
        • Hermanson G.T.
        • Mallia A.K.
        • Gartner F.H.
        • Provenzano M.D.
        • Fujimoto E.K.
        • Goeke N.M.
        • Olson B.J.
        • Klenk D.C.
        Anal. Biochem. 1985; 150: 76-85
        • Lorenz H.
        • Hailey D.W.
        • Lippincott-Schwartz J.
        Nat. Methods. 2006; 3: 205-210
        • Ruddy D.A.
        • Kronmal G.S.
        • Lee V.K.
        • Mintier G.A.
        • Quintana L.
        • Domingo Jr., R.
        • Meyer N.C.
        • Irrinki A.
        • McClelland E.E.
        • Fullan A.
        • Mapa F.A.
        • Moore T.
        • Thomas W.
        • Loeb D.B.
        • Harmon C.
        • Tsuchihashi Z.
        • Wolff R.K.
        • Schatzman R.C.
        • Feder J.N.
        Genome Res. 1997; 7: 441-456
        • Meyer M.
        • Gaudieri S.
        • Rhodes D.A.
        • Trowsdale J.
        Tissue Antigens. 2003; 61: 63-71
        • Enroth C.
        • Eger B.T.
        • Okamoto K.
        • Nishino T.
        • Nishino T.
        • Pai E.F.
        Proc. Natl. Acad. Sci. U.S.A. 2000; 97: 10723-10728
        • Lim K.
        • Ho J.X.
        • Keeling K.
        • Gilliland G.L.
        • Ji X.
        • Rüker F.
        • Carter D.C.
        Protein Sci. 1994; 3: 2233-2244
        • Masters S.L.
        • Yao S.
        • Willson T.A.
        • Zhang J.G.
        • Palmer K.R.
        • Smith B.J.
        • Babon J.J.
        • Nicola N.A.
        • Norton R.S.
        • Nicholson S.E.
        Nat. Struct. Mol. Biol. 2006; 13: 77-84
        • Henry J.
        • Mather I.H.
        • McDermott M.F.
        • Pontarotti P.
        Mol. Biol. Evol. 1998; 15: 1696-1705
        • Laing K.J.
        • Purcell M.K.
        • Winton J.R.
        • Hansen J.D.
        BMC Evol. Biol. 2008; 8: 42
        • Heid H.W.
        • Winter S.
        • Bruder G.
        • Keenan T.W.
        • Jarasch E.D.
        Biochim. Biophys. Acta. 1983; 728: 228-238
        • Mather I.H.
        Neville M.C. Daniel C.W. The Mammary Gland: Development, Regulation and Function. Plenum Publishing Corp., New York1987: 217-267
        • McManaman J.L.
        • Russell T.D.
        • Schaack J.
        • Orlicky D.J.
        • Robenek H.
        J Mammary Gland Biol. Neoplasia. 2007; 12: 259-268
        • Buchheim W.
        Naturwissenschaften. 1982; 69: 505-507
        • Wooding F.B.P.
        Symp. Zool. Soc. London. 1977; 41: 1-41
        • McManaman J.L.
        • Palmer C.A.
        • Wright R.M.
        • Neville M.C.
        J. Physiol. 2002; 545: 567-579
        • Adachi T.
        • Fukushima T.
        • Usami Y.
        • Hirano K.
        Biochem. J. 1993; 289: 523-527
        • Keon B.H.
        • Ankrapp D.P.
        • Keenan T.W.
        Biochim. Biophys. Acta. 1994; 1215: 327-336
        • Pahl H.L.
        • Baeuerle P.A.
        BioEssays. 1994; 16: 497-502
        • Martin H.M.
        • Hancock J.T.
        • Salisbury V.
        • Harrison R.
        Infect. Immun. 2004; 72: 4933-4939
        • Vorbach C.
        • Harrison R.
        • Capecchi M.R.
        Trends Immunol. 2003; 24: 512-517
        • Admyre C.
        • Johansson S.M.
        • Qazi K.R.
        • Filén J.J.
        • Lahesmaa R.
        • Norman M.
        • Neve E.P.
        • Scheynius A.
        • Gabrielsson S.
        J. Immunol. 2007; 179: 1969-1978