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Folding of Human Intestinal Lactase-phlorizin Hydrolase *

  • Ralf Jacob
    Affiliations
    From the Protein Secretion Group, Institute of Microbiology, Heinrich Heine University, Universittsstrasse 1, D-40225 Dsseldorf, Federal Republic of Germany
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  • Neil J. Bulleid
    Affiliations
    Division of Biological Sciences, Department of Biochemistry and Molecular Biology, University of Manchester, Oxford Road, Manchester M13 9PT, United Kingdom
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  • Hassan Y. Naim
    Correspondence
    To whom correspondence should be addressed: Protein Secretion Group, Inst. of Microbiology, Heinrich Heine University, Universittsstr. 1, Geb. 26.12, D-40225 Dsseldorf, FRG. Tel.: 49-211-311-37-33; Fax: 49-211-311-37-33/53-70.
    Affiliations
    From the Protein Secretion Group, Institute of Microbiology, Heinrich Heine University, Universittsstrasse 1, D-40225 Dsseldorf, Federal Republic of Germany
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  • Author Footnotes
    * This work was supported by grants from the Ministry for Research and Technology, Bonn, Federal Republic of Germany (to H. Y. N.), the Royal Society (to N. J. B.), the Deutscher Akademischer Austanschdienst, Bonn, and the British Council, Cologne, Federal Republic of Germany (to H. Y. N. and N. J. B.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
      The folding of human intestinal prolactase-phlorizin hydrolase (pro-LPH) has been analyzed in a cell-free transcription/translation system. In the presence of the thiol oxidant GSSG, disulfide bond formation in pro-LPH can be promoted concomitant with the binding of the molecule to a conformation-specific monoclonal anti-LPH antibody. Under these conditions, pro-LPH does not bind to the molecular chaperone BiP. In the absence of GSSG, on the other hand, pro-LPH does not bind to the monoclonal anti-LPH antibody, but can be immunoprecipitated with a polyclonal antibody that is directed against a denatured form of the enzyme. In this case, interaction of pro-LPH with immunoglobulin heavy chain binding protein can be discerned. The results demonstrate the existence of intramolecular disulfide bonds that are essential for the promotion of pro-LPH to a native conformation. Furthermore, BiP is involved in the folding events of pro-LPH.

      INTRODUCTION

      Most proteins in eukaryotic cells are synthesized in the cytosol and sorted to different cellular compartments or secreted into the exterior milieu by virtue of specific sorting signals or sorting patches contained in their primary, secondary, or tertiary structures (
      • Kornfeld S.
      • Mellman I.
      ;
      • Mostov K.
      • Apodeca G.
      • Aroeti B.
      • Okamoto C.
      ). Proteins destined for the plasma membrane, lysosomes, nuclear envelope, and endocytic and exocytic membranes possess a signal sequence, a stretch of at least 6 hydrophobic amino acids, that mediates the integration of the synthesized protein into the ER
      The abbreviations used are: ER
      endoplasmic reticulum
      LPH
      lactase-phlorizin hydrolase
      pro-LPH
      prolactase-phlorizin hydrolase
      LPHm
      mature lactase-phlorizin hydrolase
      PAGE
      polyacrylamide gel electrophoresis
      DTT
      dithiothreitol
      mAb
      monoclonal antibody
      pAb
      polyclonal antibody
      BiP
      immunoglobulin heavy chain binding protein.
      (
      • von Heijne G.
      ). Several modification reactions are initiated during entry of the extending polypeptide chain into the ER lumen and may facilitate the folding of the protein into native conformation. These include signal sequence cleavage, attachment of mannose-rich carbohydrate chains, and disulfide bond formation. In addition, a number of ER resident proteins, most notably, protein chaperones, such as BiP/GRP78 and calnexin, and enzymes, such as protein-disulfide isomerase, are thought to be crucial in protein folding, protein-protein interactions in oligomeric structures, and elimination of malfolded polypeptides (
      • Sanders S.L.
      • Whitfield K.M.
      • Vogel J.P.
      • Rose M.D.
      • Schekman R.W.
      ;
      • Ou W.J.
      • Cameron P.H.
      • Thomas D.Y.
      • Bergeron J.J.M.
      ) (for reviews, see
      • Hurtley S.M.
      • Helenius A.
      ,
      • Bulleid N.J.
      • Bassel-Duby R.S.
      • Freedman R.B.
      • Sambrook J.F.
      • Gething M.J.
      , and
      • Doms R.W.
      • Lamb R.A.
      • Rose J.K.
      • Helenius A.
      ).
      The pathways leading to the promotion of a protein to its final native configuration are still not completely defined. However, a large body of information has proposed that protein folding ensues by rapid interaction of hydrophobic residues in the polypeptide chain, formation of secondary structures such as α-helices and β-sheets, and finally formation of disulfide bonds or other covalent interactions to stabilize particular regions of the protein (for a review, see
      • Gething M.J.
      • Sambrook J.
      ).
      We are interested in dissecting the molecular mechanisms implicated in the generation of transport-competent and biologically active molecules of human intestinal brush border membrane. Lactase-phlorizin hydrolase (LPH), an intestinal brush border glycoprotein responsible for the digestion of lactose in mammalian milk, is one of these molecules. LPH is synthesized in human intestinal cells as a single chain mannose-rich precursor (pro-LPH; Mr = 215,000) that acquires complex glycosylated sugars in the Golgi apparatus prior to proteolytic cleavage and targeting to the brush border membrane as mature LPH (LPHm; Mr = 160,000). The subunit structure of LPHm revealed one single polypeptide under completely denaturing conditions (
      • Naim H.Y.
      • Sterchi E.E.
      • Lentze M.J.
      ). Although the quaternary structure of LPH has not been analyzed in detail, the presence of dimeric LPH forms has been suggested (
      • Danielsen E.M.
      )
      H. Y. Naim, unpublished data.
      The complete amino acid sequence of LPH has been deduced from cDNA cloning and revealed structural features of a type I protein that is synthesized with a cleavable signal sequence and that has a transmembranous orientation (
      • Mantei N.
      • Villa M.
      • Enzler T.
      • Wacker H.
      • Boll W.
      • James P.
      • Hunziker W.
      • Semenza G.
      ). Of note is the presence of a large profragment (LPHα; 868 amino acids) at the N-terminal end that precedes brush border LPHm. Recent identification of LPHα in intestinal biopsy samples (
      • Naim H.Y.
      • Jacob R.
      • Naim H.
      • Sambrook J.F.
      • Gething M.J.H.
      ) and analysis of its possible function have led to the hypothesis that LPHα may play a crucial role in the folding of pro-LPH, perhaps as an intramolecular chaperone (
      • Oberholzer T.
      • Mantei N.
      • Semenza G.
      ;
      • Naim H.Y.
      • Jacob R.
      • Naim H.
      • Sambrook J.F.
      • Gething M.J.H.
      ). The LPHα profragment is rich in hydrophobic amino acids and contains 13 cysteine residues, while mature LPH (1061 amino acids) contains, in comparison, only 6 cysteine residues. It is not known whether these cysteine residues are involved in disulfide bond formation. In fact, analysis of pro-LPH as well as cleaved LPHm under reducing and nonreducing conditions did not reveal differences in their electrophoretic mobilities (see “Results”). However, it is possible that slight variations in the apparent molecular weights cannot be detected by SDS-PAGE analysis. In this paper, we investigated the folding of pro-LPH with particular emphasis on disulfide bond formation.

      EXPERIMENTAL PROCEDURES

       Materials

      SP6 RNA polymerase, nucleotides, nuclease-treated rabbit reticulocyte lysate, amino acids minus methionine, RNasin ribonuclease inhibitor, and dog pancreas microsomal membranes were purchased from Promega. Restriction enzymes, proteinase K, and endoglycosidase H were purchased from Boehringer Mannheim. trans-[35S]Methionine was purchased from ICN.

       Cell-free Transcription

      The vector pGEM-4Z containing the complete cDNA for human LPH, pLPH (
      • Naim H.Y.
      • Lacey S.
      • Sambrook J.F.
      • Gething M.J.H.
      ), was linearized with SalI. Transcription was initiated with SP6 RNA polymerase and was carried out essentially as described (
      • Gurevich V.V.
      • Pokrovskaya I.D.
      • Obukhova T.A.
      • Zozulya S.A.
      ). Subsequently, the mixture was extracted once with phenol/chloroform and twice with chloroform. After ethanol precipitation, the RNA was resuspended in 50 μl of RNase-free water.

       Cell-free Translation

      Transcribed RNA was translated using a nuclease-treated rabbit reticulocyte lysate. The reaction mixture comprised 18 μl of reticulocyte lysate, 1 μl of 1 mM amino acids (minus methionine), 15 μCi of L-[35S]methionine, and 3 μl of transcribed RNA. Where indicated, the samples were supplemented with 1 μl of nuclease-treated microsomal membranes. Translations were performed in the absence or presence of GSSG at 30°C for 60 min.

       Reduction and Alkylation of Solubilized Biopsy Specimens

      Human small intestinal mucosas (~5-10 mg, wet weight) were obtained from patients biopsied for diagnostic purposes. They appeared normal when examined by light microscopy and expressed normal levels of brush border disaccharidase activities. Biosynthetic labeling was performed according to
      • Naim H.Y.
      • Sterchi E.E.
      • Lentze M.J.
      . Briefly, the biopsy specimens were placed on stainless steel grids in organ culture dishes, washed three times in methionine-free RPMI 1640 medium containing 10% dialyzed fetal calf serum, and incubated in the same medium for 2 h at 37°C in a CO2+ O2 (5:95, v/v) incubator. The biopsy samples were labeled with trans-[35S]methionine at 100 μCi/biopsy sample. After labeling, the specimens were washed three times in RPMI 1640 medium and homogenized at 4°C with a Teflon-glass homogenizer in 1 ml of 25 mM Tris-HCl, pH 8.1, supplemented with 50 mM NaCl and a mixture of protease inhibitors (1 mM phenylmethylsulfonyl fluoride, 1 μg/ml pepstatin, 5 μg/ml leupeptin, 1 μg/ml aprotinin, and 17.4 μg/ml benzamidine). Thereafter, the homogenates were solubilized with 1% Triton X-100 for 1 h at 4°C and centrifuged at 100,000 × g for 1 h at 4°C. The supernatant was treated with 20 mM dithiothreitol at 37°C for 2 h, cooled on ice, and alkylated with 80 mM iodoacetamide for 4 h at 4°C. The reduced and alkylated lysates were then dialyzed in the cold against 25 mM Tris-HCl, pH 8.1, containing 50 mM NaCl and 1% Triton X-100. Usually, 1 ml of the lysates was dialyzed against 500 ml of dialysis buffer, which was changed four times within 48 h. As a control, labeled biopsy specimens were processed similarly as described above, except that no DTT or iodoacetamide was added. Reduced and alkylated lysates as well as untreated lysates were immunoprecipitated with anti-LPH mAb or anti-LPH pAb. Control immunoprecipitations were performed with monoclonal antibody against intestinal sucrase-isomaltase of the brush border membrane.

       Immunoprecipitation

      Mouse monoclonal anti-human LPH antibody (anti-LPH mAb) was generously provided by Dr. Hans-Peter Hauri (Biocenter, Basel, Switzerland) (
      • Hauri H.-P.
      • Sterchi E.E.
      • Bienz D.
      • Fransen J.A.M.
      • Marxer A.
      ). Polyclonal anti-LPH antibody (anti-LPH pAb) was prepared by intramuscular injection of electrophoretically purified LPH into rabbits (
      • Naim H.Y.
      • Jacob R.
      • Naim H.
      • Sambrook J.F.
      • Gething M.J.H.
      ). The specificity of the antibody was examined by Western blotting using whole brush border membranes as an antigen (see Fig. 4, lane 1). Monoclonal anti-BiP antibody was a generous gift from Dr. Linda Hendershot (St. Jude Children's Hospital, Memphis, TN) (
      • Bole D.G.
      • Hendershot L.M.
      • Kearney J.F.
      ). To deplete the translation mixtures of ATP for coprecipitation experiments with anti-BiP antibody, the samples were incubated for 2 min at 37°C with apyrase (100 units/ml). Prior to immunoprecipitations, the antibodies were conjugated to protein A-Sepharose beads as follows. 1 μl of anti-LPH mAb, 1 μl of anti-BiP antibody, or 5 μl of anti-LPH pAb was mixed with 30 μl of protein A-Sepharose and 1 ml of sodium phosphate buffer, pH 8.0, for 2 h at 4°C. Thereafter, the beads were washed once with sodium phosphate buffer and used in the immunoprecipitations. The translation mixtures were diluted in 50 mM Tris-HCl, pH 7.4, containing 150 mM NaCl, 5 mM EDTA, 0.25% gelatin, 0.05% Nonidet P-40, and 0.02% sodium azide and immunoprecipitated with the corresponding Immunobeads for 6 h at 4°C. To study the ATP dependence of the binding of LPH to BiP, ATP (1 mM) was included in the immunoprecipitation buffer. The pelleted Sepharose beads were washed three times in dilution buffer and resuspended in 40 μl of SDS-PAGE sample buffer (62.5 mM Tris-HCl, pH 6.8, 2% (w/v) SDS, 10% (w/v) glycerol, and bromphenol blue).
      Figure thumbnail gr4
      Figure 4:Effect of reduction and alkylation on the binding capacity of anti-LPH mAb to LPH. Biopsy samples were biosynthetically labeled for 6 h with [35S]methionine, homogenized, and solubilized with Triton X-100. The detergent extracts were treated for 2 h at 37°C with 20 mM DTT and alkylated for 4 h at 4°C with iodoacetamide, and excess DTT and iodoacetamide were removed by dialysis. Reduced and alkylated lysates as well as untreated lysates were immunoprecipitated with anti-LPH mAb (lanes 2 and 3) or anti-LPH pAb (lanes 4 and 5). Immunoprecipitation with a monoclonal anti-sucrase-isomaltase antibody (Mab anti-SI) served as a control (lanes 6 and 7). Lane 1 is a Western blot (W.B.) of brush border membranes with anti-LPH pAb to confirm the specificity of this antibody in recognizing denatured forms of brush border LPH (Mr = 160,000). SIh, mannose-rich sucrase-isomaltase; SIc, complex glycosylated sucrase-isomaltase.
      In some experiments, trypsin treatment of immunoprecipitated pro-LPH was performed. Here, the immunoprecipitates were washed three times with dilution buffer and twice with phosphate-buffered saline. The beads were then resuspended in 40 μl of phosphate-buffered saline and treated with 50 μg of trypsin at 37°C for 30 min. The reaction was arrested by the addition of SDS-PAGE sample buffer and boiling for 5 min. The samples were finally analyzed by SDS-PAGE.

       Other Methods

      Highly purified brush border membranes were prepared, solubilized, and immunoprecipitated with monoclonal anti-human LPH antibody as described by
      • Naim H.Y.
      • Sterchi E.E.
      • Lentze M.J.
      . Endoglycosidase H treatment of immunoprecipitates or translation products was performed as described (
      • Naim H.Y.
      • Sterchi E.E.
      • Lentze M.J.
      ).
      Microsomal membranes were isolated as described by
      • Bulleid N.J.
      • Freedman R.B.
      . Translocation of the translated proteins was assessed by proteinase K treatment as described previously (
      • Bulleid N.J.
      • Freedman R.B.
      .

       SDS-PAGE

      Samples were prepared for electrophoresis by mixing with 5 volumes of SDS-PAGE sample buffer in the presence of 50 mM DTT for reducing conditions and in the presence of 100 mM iodoacetamide for nonreducing conditions and boiled for 5 min. SDS-PAGE was performed by the method of
      • Laemmli U.K.
      . After electrophoresis, gels were processed for autoradiography and exposed to Kodak X-Omat AR film.

      RESULTS

       Electrophoretic Analysis of Human Intestinal LPH

      To examine the presence of intermolecular or intramolecular disulfide bonds in brush border LPH, brush border membranes were solubilized and immunoprecipitated with monoclonal anti-LPH antibodies. Fig. 1 demonstrates that under completely denaturing conditions, i.e. in the presence of SDS and DTT, brush border LPH is composed predominantly of one single polypeptide of Mr = 160,000 (lane 2). When DTT was omitted from the electrophoresis sample buffer, the same protein band pattern was obtained (Fig. 1, lane 1). In addition, a high molecular weight band (Mr > 202,000, the molecular weight of the standard protein myosin) was detected. This band has been also previously detected and suggested to correspond to dimeric forms of brush border LPH (
      • Sterchi E.E.
      • Mills P.R.
      • Fransen J.A.M.
      • Hauri H.-P.
      • Lentze M.J.
      • Naim H.Y.
      • Ginsel L.
      • Bond J.
      ). Since this band is mainly detected in the nonreduced sample, we conclude that it represents dimers that are covalently associated by intermolecular disulfide bonds. However, the extent of such dimers in total brush border LPH is very low since the Mr = 160,000 polypeptide is the main LPH species detected under reducing and nonreducing conditions. Furthermore, we were not able to detect shifts in the mobilities of the brush border LPH species under reducing and nonreducing conditions. A shift in the mobility would be indicative of the presence of intramolecular disulfide bonds. It is therefore obvious that the presence of possibly existing disulfide bonds in brush border LPH cannot be assessed by this approach.
      Figure thumbnail gr1
      Figure 1:Electrophoretic analysis of human intestinal brush border LPH. Brush border membranes were purified from human small intestinal mucosas and solubilized with Triton X-100 and deoxycholate (0.5% each). The supernatant after a 100,000 × g centrifugation was immunoprecipitated with monoclonal anti-LPH antibody. The immunoprecipitates were analyzed by SDS-PAGE on 5% slab gels under reducing (lane 2) or nonreducing (lane 1) conditions. The gel was stained with Coomassie Blue. Apparent molecular masses of standard proteins (lane 3) are indicated on the right.

       Cell-free Synthesis of Pro-LPH

      In view of the results shown above, we sought to investigate the presence of disulfide bonds by utilizing a cell-free transcription/translation system. Here, a full-length LPH cDNA containing the coding sequence for human LPH in the pGEM-4Z vector (denoted pF1; see
      • Naim H.Y.
      • Lacey S.
      • Sambrook J.F.
      • Gething M.J.H.
      ) was transcribed with phage SP6 RNA polymerase. The resulting RNA was translated in a cell-free translation/translocation system consisting of rabbit reticulocyte lysate supplemented with dog pancreas microsomal vesicles (Fig. 2A). The major polypeptide synthesized in the absence of added microsomal vesicles had an apparent Mr of 200,000 (Fig. 2A, lane 1), consistent with the molecular weight of prepro-LPH calculated from the deduced amino acid sequence (
      • Mantei N.
      • Villa M.
      • Enzler T.
      • Wacker H.
      • Boll W.
      • James P.
      • Hunziker W.
      • Semenza G.
      ). In the presence of microsomal membranes, an additional polypeptide of Mr = 215,000 was generated (Fig. 2A, lane 2). To assess whether the 215-kDa species was translocated into the interior of the microsomal membranes, two different approaches were carried out. In the first approach, the microsomal membranes were separated from the reticulocyte lysates by centrifugation through a sucrose cushion. As shown in Fig. 2B (lane 2), the 215-kDa species was the predominantly labeled polypeptide in the microsomal membranes. Furthermore, the 215-kDa polypeptide was glycosylated, and its glycosylation was of the mannose-rich type since it was converted by endoglycosidase H treatment to a band of 200 kDa (Fig. 2B, lane 3), similar to the apparent molecular mass of the nascent polypeptide (lane 1). The recovery of a glycosylated 215-kDa polypeptide in the microsomal membranes indicates that the 200-kDa translation product was exposed to the glycosylation machinery in the interior of the microsomal membranes, leading to the generation of the 215-kDa species.
      Figure thumbnail gr2
      Figure 2:A, cell-free translation of LPH mRNA in the presence or absence of microsomal membranes. Transcription of the full-length human LPH cDNA was initiated with SP6 RNA polymerase. Transcribed RNA was translated using a nuclease-treated rabbit reticulocyte lysate containing L-[35S]methionine. Translation was performed in the absence (lane 1) or presence (lane 2) of microsomal membranes (MM). The samples were analyzed by SDS-PAGE on 7% slab gels and autoradiography. B, endoglycosidase H (Endo H) treatment of LPH synthesized in a cell-free system. LPH was synthesized in the presence (lanes 2 and 3) or absence (lane 1) of microsomal membranes. The microsomal membranes were isolated by centrifugation through a sucrose cushion (lanes 2 and 3) and treated with endoglycosidase H (lane 3) or were not treated (lane 2). The samples were finally analyzed by SDS-PAGE on 7% slab gels followed by autoradiography. C, treatment of translation products with proteinase K. Translation of LPH was carried out as described for A. Parts of the LPH translation products in the presence (lanes 1 and 2) or absence (lanes 3 and 4) of microsomal membranes were treated with proteinase K (Prot. K) (lanes 2 and 4). The samples were analyzed by SDS-PAGE on 7% slab gels and autoradiography. D, treatment of translation products with proteinase K in the presence or absence of Triton X-100. Translation of LPH was carried out as described for A. The LPH translation products in the presence of microsomal membranes (lanes 2 and 3) were treated with proteinase K after solubilization with Triton X-100 (TX-100) (lane 3) or without prior solubilization (lane 2). The samples were analyzed by SDS-PAGE on 7% slab gels and autoradiography. Lane 1 represents LPH synthesized in the absence of microsomal membranes and without treatment with Triton X-100 or proteinase K.
      To further assess the translocation of the translated pro-LPH products across the microsomal membranes, the susceptibility of the nascent 200-kDa polypeptide and its glycosylated 215-kDa counterpart to proteinase K was examined. As depicted in Fig. 2C, only the nonglycosylated 200-kDa polypeptide was degraded by this treatment, while glycosylated 215-kDa pro-LPH remained unaffected (lane 2). Treatment of the translated product alone (Fig. 2C, lane 3) with proteinase K resulted also in a degradation of this polypeptide (Fig. 2C, lane 4). On the other hand, solubilization of the microsomal membranes with Triton X-100 followed by proteinase K treatment resulted in a complete degradation of the 215-kDa species (Fig. 2D, lane 3). Therefore, glycosylated 215-kDa pro-LPH was not accessible to proteinase K since it was found in the interior of the microsomal vesicles; solubilization of the membranes led to exposure of this species and consequently to its degradation by proteinase K. Altogether, the data obtained clearly demonstrate that the 200-kDa species is translocated into the microsomal membranes.

       Formation of Disulfide Bonds Is an Essential Event in the Folding of Pro-LPH

      To investigate the presence of cotranslationally formed disulfide bonds, the translation reaction was carried out in the presence of GSSG. GSSG oxidizes existing thiol groups and therefore facilitates the formation of disulfide bonds. It was required in the reaction mixture to compensate for the presence of the reducing agent dithiothreitol in the commercially prepared reticulocyte lysates. Varying concentrations of GSSG were added to the reticulocyte lysates before initiation of translation to obtain a lysate that was competent in forming disulfide bonds in the newly synthesized protein. A change in the thiol/disulfide redox status of the translation products has been shown to influence the migration pattern of many proteins (
      • Goldenberg D.P.
      • Creighton T.E.
      ). However, this could not be assessed electrophoretically for brush border LPHm (Fig. 1), LPH isolated from biopsy samples (see Fig. 4), or LPH synthesized in a cell-free system (data not shown). We therefore sought to determine at the immunochemical level whether GSSG induced conformational alterations in pro-LPH reminiscent of the formation of native disulfide bonds. Here, we used two antibodies directed against LPH: a conformation-specific monoclonal anti-LPH antibody (anti-LPH mAb) (
      • Hauri H.-P.
      • Sterchi E.E.
      • Bienz D.
      • Fransen J.A.M.
      • Marxer A.
      ) and a polyclonal antibody directed against the denatured form of LPH (anti-LPH pAb). In the absence of GSSG, immunoprecipitation of the translation products with anti-LPH mAb did not reveal molecular species corresponding to pro-LPH (Fig. 3A, lane 1), although the translation products contained pro-LPH molecules (Fig. 3B, lane 1). In fact, anti-LPH pAb clearly recognized pro-LPH species that were translated in the presence or absence of GSSG (Fig. 3B). On the other hand, in the presence of GSSG, a dose-dependent reactivity of pro-LPH with anti-LPH mAb was manifested. The intensity of pro-LPH (Mr = 215,000) increased when the concentration of GSSG was raised from 1 mM (Fig. 3A, lane 2) to 2 mM (lane 3), but decreased at higher concentrations (up to 4 mM) (lanes 4 and 5). Comparison of the labeling intensities by scanning densitometry of immunoprecipitated pro-LPH (Fig. 3A) and total pro-LPH in the translation products (data not shown) revealed that the decrease in the intensity of immunoprecipitated pro-LPH was due to inhibition of protein synthesis by GSSG and not due to unfolding of pro-LPH. In fact, Fig. 3D demonstrates that the ratio of immunoprecipitated pro-LPH at a certain GSSG concentration versus total synthesized pro-LPH at the same GSSG concentration increased when the concentration of GSSG was raised from 1 to 2 mM, but reached a plateau at higher GSSG concentrations. This result therefore indicates that anti-LPH mAb immunoprecipitates similar proportions of pro-LPH from total pro-LPH synthesized in the presence of 2, 3, and 4 mM GSSG. This strongly suggests that the epitope recognized by the mAb in the pro-LPH molecule does not undergo significant conformational alterations at GSSG concentrations between 2 and 4 mM.
      Figure thumbnail gr3
      Figure 3:Cell-free translation of LPH mRNA in the presence of GSSG. A, immunoprecipitation of pro-LPH synthesized in the presence of increasing concentrations of GSSG. Translation of pro-LPH was performed in the presence of microsomal membranes as described for A and in the presence of varying concentrations of the oxidant GSSG as indicated. The translation products were immunoprecipitated with anti-LPH mAb and analyzed by SDS-PAGE on 7% slab gels followed by autoradiography. B, immunoprecipitation of pro-LPH with anti-LPH pAb. Products of translation in the presence of microsomal membranes and in the absence (lane 1) or presence (lane 2) of 2 mM GSSG were immunoprecipitated with anti-LPH pAb and resolved by SDS-PAGE on 7% slab gels followed by autoradiography. C, immunoprecipitation of pro-LPH in the presence of GSSG. Biopsy samples were biosynthetically labeled for 2 h and solubilized. The detergent extracts were treated with varying concentrations of GSSG as indicated and immunoprecipitated with anti-LPH mAb. The immunoprecipitates were analyzed by SDS-PAGE on 7% slab gels. D, comparison of immunoprecipitated pro-LPH with total synthesized pro-LPH at different concentrations of GSSG. The intensity of immunoprecipitated pro-LPH at different GSSG concentrations (A) was compared with that of total pro-LPH synthesized in the presence of similar GSSG concentrations.
      On the other hand, it is possible that GSSG may influence the binding of the mAb to the pro-LPH species at increasing concentrations of GSSG, thus leading to reduced amounts of immunoprecipitated pro-LPH at concentrations higher than 2 mM. To investigate this issue, we labeled biopsy samples biosynthetically for 2 h, after which time only the 215-kDa mannose-rich pro-LPH species becomes labeled (
      • Naim H.Y.
      • Sterchi E.E.
      • Lentze M.J.
      ;
      • Naim H.Y.
      ). The detergent extracts of the biopsy samples were then treated with various concentrations of GSSG and immunoprecipitated with anti-LPH mAb. As shown in Fig. 3C, essentially similar amounts of labeled pro-LPH were immunoprecipitated at increasing concentrations of GSSG. Therefore, this result demonstrates that GSSG per se does not affect antibody-antigen binding. Consequently, these and the scanning data are consistent with the view that the decrease in the labeling intensity of immunoprecipitated pro-LPH (Fig. 3A) is due to reduced amounts of translated pro-LPH at high GSSG concentrations. Taken together, the reactivity of the conformation-specific antibody with pro-LPH generated in the presence of GSSG, but not in its absence, strongly suggests that the oxidant facilitates the folding of pro-LPH by formation of disulfide bonds, whereby pro-LPH ultimately assumes a conformation similar to that of native intestinal pro-LPH.

       The Epitope Recognized by the Monoclonal Antibody HBB 1/909 Contains Disulfide Bonds

      The observation that anti-LPH mAb (HBB 1/909) binds LPH forms obtained in the presence of GSSG suggests that the epitope recognized by the antibody contains disulfide bonds. To examine this possibility, it was necessary to reduce possibly existing disulfide bonds in LPH and subject the reduced forms to immunoprecipitation with anti-LPH mAb. For this purpose, biopsy samples were biosynthetically labeled for 6 h, after which time the mannose-rich LPH precursor polypeptide, pro-LPH (215 kDa), as well as intracellularly cleaved LPHm (160 kDa) can be detected (
      • Naim H.Y.
      • Sterchi E.E.
      • Lentze M.J.
      ;
      • Naim H.Y.
      ). The biopsy lysates were then treated with DTT, alkylated with iodoacetamide, dialyzed to remove any residual reagents, and finally immunoprecipitated with anti-LPH mAb (HBB 1/909) or with anti-LPH pAb. Fig. 4(lane 2) demonstrates that mannose-rich and complex glycosylated precursor pro-LPH as well as LPHm (160 kDa) (
      • Naim H.Y.
      • Sterchi E.E.
      • Lentze M.J.
      ;
      • Naim H.Y.
      ) were immunoprecipitated with anti-LPH mAb (HBB 1/909) in the absence of DTT treatment. By contrast, when the biopsy lysates were treated with DTT prior to immunoprecipitation, no bands corresponding to LPH molecules were detected (Fig. 4, lane 3). Similar lysates were immunoprecipitated with anti-LPH pAb. This antibody reacts with the denatured 160-kDa LPHm species on Western blots (Fig. 4, lane 1). Under native conditions, anti-LPH pAb recognized only the mannose-rich species (Fig. 4, lane 4). The reactivity of this antibody with LPH forms increased substantially upon reduction of the biopsy lysates. As shown in Fig. 4(lane 5), the intensity of the immunoprecipitated mannose-rich pro-LPH increased by almost a 4-fold factor in comparison with that obtained under native conditions (lane 4). In addition, the 160-kDa LPHm species was also immunoprecipitated with anti-LPH pAb, although to a lesser extent than with anti-LPH mAb under native conditions (Fig. 4, lane 2). Since anti-LPH pAb recognizes the denatured form of LPH and since its binding to the mannose-rich species increases upon reduction of the lysates with DTT, we conclude that the mannose-rich form of pro-LPH recognized by anti-LPH pAb under native conditions corresponds to an unfolded or intermediate folded form of pro-LPH. On the other hand, anti-LPH mAb (HBB 1/909) binds native LPH and fails to precipitate LPH that has been reduced with DTT, strongly suggesting that the epitope recognized by this antibody contains disulfide bonds. It should be mentioned that no change in the electrophoretic mobilities of reduced and alkylated LPH species (Fig. 4, lane 5) relative to the nonreduced species (lane 2) was detected. This is consistent with the notion that an electrophoretic assessment of disulfide bonds in LPH (Fig. 1) is not possible.
      The immunoprecipitation of LPH forms from reduced lysates with anti-LPH pAb indicated that LPH in these lysates did not undergo substantial degradation owing to the reduction procedure. To corroborate these results and to determine whether other proteins are still intact in the reduced and alkylated lysates, these lysates were immunoprecipitated with a monoclonal antibody directed against sucrase-isomaltase (Fig. 4, Mab anti-SI), a major brush border membrane glycoprotein. As shown in Fig. 4, the mannose-rich (SIh) as well as complex glycosylated (SIc) sucrase-isomaltase bands (see
      • Naim H.Y.
      • Sterchi E.E.
      • Lentze M.J.
      ) were revealed in both the DTT-treated (lane 7) and untreated (lane 6) samples. Moreover, the labeling intensity of these forms did not change. The data show that the integrity of the solubilized intestinal proteins in the biopsy did not change drastically as a consequence of reduction and alkylation. Moreover, the epitope recognized by the monoclonal sucrase-isomaltase antibody appears not to involve disulfide bonds, in contrast to anti-LPH mAb. In essence, the data indicate that under nondenaturing conditions, anti-LPH mAb recognizes an epitope in native LPH in which disulfide bond(s) are essential.

       Binding of Pro-LPH to BiP

      In addition to enzymes involved in protein folding, such as protein-disulfide isomerase, a number of other ER resident proteins, known as molecular chaperones, function in vivo as stabilizers of partially folded intermediates of proteins. We wanted to determine whether partially folded or unfolded intermediates of pro-LPH interact with BiP, a well characterized molecular chaperone (
      • Haas I.G.
      • Wabl M.R.
      ). For this purpose, pro-LPH was translated and translocated into the microsomal membranes. The reaction was performed in the presence or absence of GSSG. Furthermore, apyrase was added to the samples to deplete them of ATP since hydrolysis of ATP bound to BiP may result in the release of the associated protein (
      • Munro S.
      • Pelham H.R.B.
      ;
      • Kassenbrock C.K.
      • Kelly R.B.
      ;
      • Flynn G.C.
      • Chappell T.G.
      • Rothman J.E.
      ). As shown in Fig. 5, immunoprecipitation of the membranes containing the translation products with anti-BiP antibody revealed a glycosylated pro-LPH band when the reaction was performed in the absence of GSSG (lane 1), but not its presence (lane 2). Since GSSG promotes the formation of disulfide bonds and hence facilitates the generation of a native pro-LPH conformation (see Fig. 3), we conclude that only unfolded or partially folded pro-LPH structures bind BiP.
      Figure thumbnail gr5
      Figure 5:BiP binds pro-LPH in an ATP-dependent manner. Translations were carried out as described for A either in the presence (lanes 2 and 4) or absence (lanes 1 and 3) of added GSSG (2 mM). Translations either were depleted of ATP by incubation with apyrase for 2 min at 37°C (lanes 1 and 2) prior to immunoprecipitation or were not treated with apyrase and incubated with ATP (1 mM) present in the immunoprecipitation buffer (lanes 3 and 4). All samples were immunoprecipitated with anti-BiP. The immunoprecipitates were resolved by SDS-PAGE on 7% slab gels and submitted to fluorography.
      We also demonstrated that the complex formed between pro-LPH and BiP can be dissociated in the presence of ATP. Pro-LPH translation products synthesized in the presence or absence of GSSG were immunoprecipitated with antibodies to BiP either in the presence or absence of 1 mM ATP present in the immunoprecipitation buffer. The results shown in Fig. 5 reveal that coimmunoprecipitation of pro-LPH occurs only when GSSG and ATP are absent from the translation reactions (lane 1) and that this complex can be dissociated in the presence of ATP (lane 3). This shows that the association of BiP with pro-LPH is a specific interaction and confirms previous results that demonstrate the ATP-dependent interaction of BiP with malfolded proteins (
      • Hurtley S.M.
      • Helenius A.
      ).
      To provide further evidence that the conformation of pro-LPH bound to BiP is different from that generated in the presence of 2 mM GSSG, we performed protease sensitivity assays. It is known that native pro-LPH undergoes intracellular cleavage to the brush border membrane LPHm species (
      • Danielsen E.M.
      • Skovbjerg H.
      • Norn O.
      • Sjstrm H.
      ;
      • Hauri H.-P.
      • Sterchi E.E.
      • Bienz D.
      • Fransen J.A.M.
      • Marxer A.
      ;
      • Naim H.Y.
      • Sterchi E.E.
      • Lentze M.J.
      ;
      • Naim H.Y.
      ). Furthermore, the site of cleavage has been proposed to lie between Arg868 and Ala869 (
      • Mantei N.
      • Villa M.
      • Enzler T.
      • Wacker H.
      • Boll W.
      • James P.
      • Hunziker W.
      • Semenza G.
      ;
      • Montgomery R.K.
      • Bller H.A.
      • Rings E.H.H.M.
      • Grand R.J.
      ) and therefore represents a potential trypsin cleavage site. In fact, treatment of recombinant pro-LPH expressed in COS-1 cells with trypsin generates a brush border-like species (
      • Naim H.Y.
      • Lacey S.
      • Sambrook J.F.
      • Gething M.J.H.
      ). It is therefore possible that a correctly folded pro-LPH molecule should be cleaved by trypsin to the mannose-rich analogue of LPHm, while malfolded pro-LPH should reveal a different behavior toward trypsin. For this purpose, pro-LPH species that bind or do not bind BiP were translated in the absence or presence of GSSG, respectively, immunoprecipitated with anti-LPH pAb or anti-LPH mAb, and finally treated with trypsin. As expected, anti-LPH pAb precipitated the nascent 200-kDa polypeptide as well as the glycosylated 215-kDa species (Fig. 6, lane 1). On the other hand, anti-LPH mAb reacted exclusively with the glycosylated species generated in the presence of GSSG (lane 3). Trypsin treatment of pro-LPH translated in the absence of GSSG and immunoprecipitated with the pAb resulted in a complete degradation of this species (Fig. 6, lane 2). By contrast, pro-LPH immunoprecipitated with anti-LPH mAb was cleaved into two polypeptides in the presence of trypsin, a 135-kDa species and an ~80-90-kDa polypeptide (Fig. 6, lane 4). The variations in the reactivity with trypsin corroborate the findings that GSSG assists the formation of a native conformation of pro-LPH, while in its absence, a malfolded species that binds to BiP is generated.
      Figure thumbnail gr6
      Figure 6:Trypsin sensitivity assays. Pro-LPH was synthesized in the presence of microsomal membranes and in the absence (lanes 1 and 2) or presence (lanes 3 and 4) of 2 mM GSSG. The translation products were immunoprecipitated with anti-LPH pAb (lanes 1 and 2) or anti-LPH mAb (lanes 3 and 4). The immunoprecipitates (IP) were treated with trypsin (lanes 2 and 4) or were not treated (lanes 1 and 3). The samples were finally prepared for SDS-PAGE analysis on 7% slab gels.

      DISCUSSION

      In this paper, we analyzed the folding state of pro-LPH in a cell-free transcription/translation system with particular emphasis on the formation of intramolecular disulfide bonds. The pro-LPH molecule contains 19 cysteine residues, 13 of which are found in a large domain at the N-terminal end (~45% of total pro-LPH) that is cleaved off during maturation of pro-LPH to LPHm (
      • Mantei N.
      • Villa M.
      • Enzler T.
      • Wacker H.
      • Boll W.
      • James P.
      • Hunziker W.
      • Semenza G.
      ). Ample evidence has accumulated to suggest that the native conformation of proteins is promoted by the formation of disulfide bonds that stabilize particular structures within the protein (
      • Creighton T.E.
      ,
      • Creighton T.E.
      ). It is therefore important to determine whether disulfide bond formation is an essential event in the folding process of pro-LPH. While for many proteins the presence of disulfide bonds could be manifested by shifts in the protein mobility on SDS-PAGE under reducing conditions (
      • Goldenberg D.P.
      • Creighton T.E.
      ), this approach may not be adequately sensitive when large proteins such as pro-LPH or LPHm are studied. In fact, the initial analysis of the subunit structure of pro-LPH and LPHm by SDS-PAGE under reducing or nonreducing conditions did not provide any evidence of the presence of intramolecular disulfide bridges since no major electrophoretic alterations of reduced versus nonreduced components could be discerned. This is even more difficult to detect, if at all, if only a few cysteine residues are implicated in disulfide bond formation. We therefore investigated a possible role of disulfide bond formation in the folding of pro-LPH using a rabbit reticulocyte lysate system supplemented with microsomal membranes derived from dog pancreas. One of the advantages of this system is that disulfide bond formation can be promoted and controlled by the addition of the thiol oxidant GSSG. The promotion of a native conformation in which disulfide bonds are implicated can then be monitored by the binding capacity of pro-LPH to a conformation-specific monoclonal antibody. However, this requires that the epitope recognized by this monoclonal antibody contains disulfide bonds.
      In intestinal biopsy samples, LPH is synthesized as a mannose-rich polypeptide (pro-LPH) that undergoes intracellular cleavage to LPHm. Our data demonstrate that anti-LPH mAb (HBB1/909) recognizes the pro-LPH and LPHm species under conditions that do not disrupt the native conformation of LPH forms. When the native conformation of pro-LPH and LPHm is altered upon reduction of the biopsy lysates with DTT, anti-LPH mAb fails to bind pro-LPH and LPHm. By contrast, a control polyclonal antibody, which binds denatured forms of LPH on Western blots, immunoprecipitates the LPHm species only after the biopsy lysates have been reduced with DTT. Moreover, the binding capacity of this antibody to pro-LPH increases substantially upon DTT reduction, most likely due to unfolding of pro-LPH. These findings indicate that anti-LPH mAb (HBB 1/909) specifically binds regions in pro-LPH that implicate disulfide bonds in their secondary structure, while the polyclonal antibody binds the unfolded or intermediate forms of LPH. This underscores, on the other hand, the important role played by disulfide bonds in the generation of a native conformation of LPH.
      The translated nascent polypeptide chain of pro-LPH as well as its glycosylated form obtained in the presence of microsomal membranes reveal similar apparent molecular weights compared with their counterparts in intestinal cells. In the absence of GSSG, these polypeptides reacted with a polyclonal anti-LPH antibody, but not with a monoclonal anti-LPH antibody, indicating that under these conditions, pro-LPH has not yet assumed its native conformation. This is further corroborated by the following two observations. (i) Pro-LPH binds the molecular chaperone BiP, which is known to stabilize unfolded or partially folded protein molecules (
      • Bulleid N.J.
      • Bassel-Duby R.S.
      • Freedman R.B.
      • Sambrook J.F.
      • Gething M.J.
      ) in an ATP-dependent manner; and (ii) pro-LPH generated in the absence of GSSG undergoes complete degradation upon trypsin treatment, while pro-LPH obtained in the presence of GSSG is cleaved to two distinct polypeptides, one of which most likely corresponds to the mannose-rich counterpart of LPHm.
      Upon addition of GSSG to microsomal membranes, a dose-dependent reactivity of pro-LPH with the conformation-specific monoclonal anti-LPH antibody can be manifested. In view of the binding specificity of anti-LPH mAb, this result is indicative of a native conformation of pro-LPH being facilitated by the formation of disulfide bonds. The failure of pro-LPH to bind BiP strongly favors the notion that pro-LPH has assumed a native conformation under these conditions.
      The profile of monoclonal anti-LPH antibody binding to pro-LPH at varying concentrations of GSSG parallels most likely the formation of disulfide bonds. This reaction appears to reach equilibrium at a GSSG concentration of 2 mM, at which the binding of pro-LPH to the monoclonal antibody is maximal. At lower GSSG concentrations, the intensity of pro-LPH immunoprecipitated with anti-LPH mAb is markedly reduced. This could be explained by the presence of folding intermediates of pro-LPH that do not efficiently bind the conformation-specific antibody. Alternatively, concentrations lower than 2 mM GSSG promote the formation of disulfide bonds of only a proportion of existing pro-LPH molecules, which ultimately bind to the monoclonal antibody. Since a protein can refold in vitro without the presence of other protein components, it is anticipated that secondary and tertiary structures other than disulfide bridges form in pro-LPH regardless of the absence or presence of GSSG. These structures, however, do not endow pro-LPH per se with a native configuration since pro-LPH does not bind the conformation-specific antibody in the absence of GSSG. At concentrations higher than the optimum, GSSG inhibits protein synthesis as shown previously (
      • Scheele G.
      • Jacoby R.
      ;
      • Marquardt T.
      • Hebert D.N.
      • Helenius A.
      ).
      Finally, it remains to be determined whether disulfide bond formation has directly affected the folding state of pro-LPH by stabilizing folding intermediates of the molecule or has secondary effects, for example by influencing the glycosylation state, which in turn alters the configuration of pro-LPH. In fact, evidence has been obtained to show that disulfide bond formation and the glycosylation state of several proteins are closely linked events (
      • Bulleid N.J.
      • Bassel-Duby R.S.
      • Freedman R.B.
      • Sambrook J.F.
      • Gething M.J.
      ).

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