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The Prosequence of Human Lactase-Phlorizin Hydrolase Modulates the Folding of the Mature Enzyme*

  • Ralf Jacob
    Affiliations
    Department of Physiological Chemistry, School of Veterinary Medicine Hannover, Bünteweg 17, Hannover D-30559, Germany
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  • Karen Peters
    Affiliations
    Department of Physiological Chemistry, School of Veterinary Medicine Hannover, Bünteweg 17, Hannover D-30559, Germany
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  • Hassan Y. Naim
    Correspondence
    To whom correspondence should be addressed. Tel.: 49-511-953-8780; Fax: 49-511-953-8585
    Affiliations
    Department of Physiological Chemistry, School of Veterinary Medicine Hannover, Bünteweg 17, Hannover D-30559, Germany
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  • Author Footnotes
    * This work was supported by Grant Na 331/1-2 from the Deutsche Forschungsgemeinschaft, Bonn, Germany and the Sonderforschungsbereich 280.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
      The efficient transport of proteins along the secretory pathway requires that the polypeptide adopts a stably folded conformation to egress the endoplasmic reticulum (ER). The transport-competent precursor of the brush border enzyme LPH, pro-LPH, undergoes an intracellular cleavage process in thetrans-Golgi network between Arg734 and Leu735 to yield LPHβinitial. The role of the prodomain comprising the N-terminally located 734 amino acids of pro-LPH, LPHα, in the folding events of LPHβinitial has been analyzed by the individual expression of both forms in COS-1 cells. Following synthesis at 37 °C LPHβinitialacquires a misfolded and enzymatically inactive conformation that is degraded by trypsin. A temperature shift to 20 °C generates a stable, trypsin-resistant, and enzymatically active LPHβinitial indicating that the individual expression of LPHβinitial results in a temperature-sensitive conformation. This form interacts at non-permissive temperatures sequentially with the ER chaperones immunoglobulin-binding protein and calnexin resulting in an ER retention. The LPHα prodomain resides in the ER when individually expressed. It reveals compact structural features that are stabilized by disulfide bridges. LPHα and LPHβinitial readily interact with each other upon coexpression, and this interaction appears to trigger the formation of a trypsin-resistant, correctly folded, enzymatically active, and transport-competent LPHβinitial polypeptide. These data clearly demonstrate that the proregion of pro-LPH is an intramolecular chaperone that is critically essential in facilitating the folding of the intermediate form LPHβinitial in the context of the pro-LPH polypeptide.
      ER
      endoplasmic reticulum
      LPH
      lactase-phlorizin hydrolase (all forms)
      pro-LPH
      uncleaved precursor of LPH
      mAb
      monoclonal antibody
      pAb
      polyclonal antibody
      pro-LPHh
      mannose-rich precursor
      pro-LPHc
      complex-glycosylated precursor
      TX-100
      Triton X-100
      TEMED
      N,N,N′,N′-tetramethylenediamine
      DMEM
      Dulbecco's modified Eagle's medium
      CFP
      cyan fluorescence protein
      YFP
      yellow fluorescence protein
      GSSG
      oxidized glutathione
      endo H
      endo-β-acetylglucosaminidase H
      endo F/GF
      endo-β-N-acetylglucosaminidase F/N-glucosidase F
      BiP
      immunoglobulin-binding protein
      The endoplasmic reticulum (ER1) is the fabric in which newly synthesized membrane-bound and secretory proteins are extensively processed and shaped to attain a competent mobility within the cell and to achieve a functional maturation. A considerable number of pivotal modification reactions commence already during the translocation of the extending polypeptide across the ER and facilitate ultimately the folding of the protein into a native conformation (for review see Ref.
      • Gething M.J.
      • Sambrook J.
      ). Examples are signal sequence cleavage from the nascent polypeptide chain (
      • von Heijne G.
      ), cotranslational core N-glycosylation at particular Asn residues belonging to the sequon (Asn-X-Ser/Thr) (
      • Welply J.K.
      • Shenbagamurthi P.
      • Lennarz W.J.
      • Naider F.
      ), disulfide bond formation (
      • Segal M.S.
      • Bye J.M.
      • Sambrook J.F.
      • Gething M.J.
      ), and transient interactions of the folding polypeptide with a set of ER resident accessory proteins, such as molecular chaperones (
      • Tatu U.
      • Helenius A.
      ,
      • Wilson R.
      • Lees J.F.
      • Bulleid N.J.
      ). In the lumen of the ER, proteins assume their secondary and tertiary structures and, in the case of multisubunit polypeptides, assemble their individual subunits into heterodimeric, homodimeric, or larger order complexes (
      • Hurtley S.M.
      • Helenius A.
      ). Essentially only correctly folded and assembled proteins can egress the ER and continue their further journey along the secretory pathway. The conformational status of newly synthesized polypeptides is monitored in the lumen of the ER by an efficient quality control mechanism (
      • Hammond C.
      • Helenius A.
      ). Proteins that fail to acquire a correct three-dimensional structure are retained in the ER and ultimately degraded, presumably in the cytosol by the ER-associated ubiquitin-proteasome pathway (
      • Ellgaard L.
      • Molinari M.
      • Helenius A.
      ). Increasing evidence points to the possible existence of a monitoring system that operates beyond the ER. A naturally occurring mutant of intestinal sucrase-isomaltase (
      • Ouwendijk J.
      • Moolenaar C.E.
      • Peters W.J.
      • Hollenberg C.P.
      • Ginsel L.A.
      • Fransen J.A.
      • Naim H.Y.
      ) or a temperature-sensitive mutant of the G protein of vesicular stomatitis virus (
      • Chen S.S.
      • Huang A.S.
      ) exit the ER but are blocked in a pre-Golgi compartment instead of transported further to their final destination, the cell surface. The acquisition of a protein to a configuration similar to that of the mature and biologically active species does not always constitute an absolute requirement for its transport along the secretory pathway to the final destination. Nevertheless, retarded transport kinetics and impaired function are frequently the consequences of altered protein folding as has been demonstrated for some mutants of the hemagglutinin of the influenza virus (
      • Tatu U.
      • Hammond C.
      • Helenius A.
      ) and deletion mutants of intestinal lactase-phlorizin hydrolase (
      • Jacob R.
      • Weiner J.R.
      • Stadge S.
      • Naim H.Y.
      ,
      • Panzer P.
      • Preuss U.
      • Joberty G.
      • Naim H.Y.
      ). Presumably, for some proteins only correct folding of particular subdomains is adequate for their mobility along the secretory pathway. It is conceivable that the structural features of the protein critically determine whether or not a protein is retained in the ER by binding specific chaperones if it did not fulfill particular folding criteria.
      Several proteins possess proregions or propeptides at the N-terminal end, which undergo cleavage during or after maturation of the precursor polypeptide. Cleavage of these propeptides is associated in many cases with biological activation of the final mature form of the protein () as in the cases of zymogens, neuropeptides, and prohormones (
      • Steiner D.F.
      • Docherty K.
      • Carroll R.
      ). An important function of propeptides is that of an intramolecular chaperone that regulates and affects the folding of the precursor protein. A well-studied example is subtilisin. The propeptide of this protein comprises 77 amino acids and is critically important for the folding of the remaining 275 amino acids that constitute the mature and active protein (
      • Zhu X.L.
      • Ohta Y.
      • Jordan F.
      • Inouye M.
      ). Human small intestinal lactase-phlorizin hydrolase (LPH, EC 3.2.1.23–62), an important component of the brush-border membrane, comprises a relatively large proregion (LPHα) that accounts for about 45% of the precursor molecule (prepro-LPH, 1927 amino acids) (
      • Mantei N.
      • Villa M.
      • Enzler T.
      • Wacker H.
      • Boll W.
      • James P.
      • Hunziker W.
      • Semenza G.
      ) and a brush-border mature polypeptide, LPHβ. Initial studies have demonstrated that LPH is synthesized as a single-chain polypeptide precursor, prepro-LPH, that undergoes two sequential intracellular cleavage steps: the first in the ER to pro-LPH (215-kDa) and the second, following terminal glycosylation in the Golgi apparatus, to a 160-kDa LPH (
      • Skovbjerg H.
      • Danielsen E.M.
      • Noren O.
      • Sjostrom H.
      ,
      • Hauri H.P.
      • Sterchi E.E.
      • Bienz D.
      • Fransen J.A.
      • Marxer A.
      ,
      • Naim H.Y.
      • Sterchi E.E.
      • Lentze M.J.
      ). Based on sequence analyses of various biosynthetic forms of LPH, recent studies have provided an unequivocal evidence for the existence of an additional cleavage step occurring extracellularly in the intestinal lumen (
      • Jacob R.
      • Radebach I.
      • Wuthrich M.
      • Grunberg J.
      • Sterchi E.E.
      • Naim H.Y.
      ,
      • Wuthrich M.
      • Grunberg J.
      • Hahn D.
      • Jacob R.
      • Radebach I.
      • Naim H.Y.
      • Sterchi E.E.
      ). Thus, the initial cleavage step of complex-glycosylated pro-LPH takes place at residues Arg734/Leu735, implicates a trypsin-like protease, and generates an LPH polypeptide encompassing residues 735–1927 (
      • Wuthrich M.
      • Grunberg J.
      • Hahn D.
      • Jacob R.
      • Radebach I.
      • Naim H.Y.
      • Sterchi E.E.
      ) that was denoted LPHβinitial (
      • Yeh K.Y.
      • Yeh M.
      • Holt P.R.
      ). The LPHβinitial molecule is subsequently targeted to the brush-border membrane where a final digestion by luminal trypsin occurs at Arg868/Ala869, which eliminates a peptide stretch from residues Leu735 to Arg868 yielding LPHβfinal (
      • Jacob R.
      • Radebach I.
      • Wuthrich M.
      • Grunberg J.
      • Sterchi E.E.
      • Naim H.Y.
      ,
      • Wuthrich M.
      • Grunberg J.
      • Hahn D.
      • Jacob R.
      • Radebach I.
      • Naim H.Y.
      • Sterchi E.E.
      ). This form is the polypeptide that exerts its enzymatic function as a β-galactosidase in the small intestine. The cleavage of pro-LPH to LPHβinitial and its potential implications on the function and trafficking on this form has been studied in heterologous transfection systems. Meanwhile it has become clear that uncleaved pro-LPH is a transport-competent (
      • Naim H.Y.
      • Lacey S.W.
      • Sambrook J.F.
      • Gething M.J.
      ), a correctly sorted (
      • Grunberg J.
      • Luginbuhl U.
      • Sterchi E.E.
      ,
      • Jacob R.
      • Brewer C.
      • Fransen J.A.
      • Naim H.Y.
      ), and an enzymatically active molecule (
      • Naim H.Y.
      • Lacey S.W.
      • Sambrook J.F.
      • Gething M.J.
      ), and hence cleavage of pro-LPH in intestinal cells is neither required for activation of the enzyme, nor is it implicated in the transport and sorting events. LPHβinitial contains all the information needed for apical sorting of pro-LPH, whereas LPHα is devoid of sorting signals despite striking sequence homologies shared with LPHβinitial (
      • Mantei N.
      • Villa M.
      • Enzler T.
      • Wacker H.
      • Boll W.
      • James P.
      • Hunziker W.
      • Semenza G.
      ). Moreover, the LPHα profragment is devoid of catalytic activity, because the lactase and phlorizin hydrolase activities have been assigned to glutamates 1271 and 1747 of LPHβfinal, respectively (
      • Wacker H.
      • Keller P.
      • Falchetto R.
      • Legler G.
      • Semenza G.
      ), and, moreover, the activities of the precursor pro-LPH and LPHβfinal are identical (
      • Naim H.Y.
      • Lacey S.W.
      • Sambrook J.F.
      • Gething M.J.
      ) for lactose or phlorizin hydrolysis.
      Based on the initial concepts that pro-LPH is immediately cleaved at the Arg868/Ala869 site a cDNA clone encoding the polypeptide Ala869-Phe1927, LPHβfinal, was expressed individually in the absence of the profragment, and its biosynthetic and structural features were analyzed. It was shown that the polypeptide generated was an enzymatically inactive protein, and most of it was retained as a mannose-rich glycosylated indicative of a predominant ER localization. This has lead to the hypothesis that the profragment LPHα functions as an intramolecular chaperone that is implicated in the folding of the LPHβ domain in the ER (
      • Oberholzer T.
      • Mantei N.
      • Semenza G.
      ,
      • Naim H.Y.
      • Jacob R.
      • Naim H.
      • Sambrook J.F.
      • Gething M.J.
      ). The intestinal form of LPHα is neither N- nor O-glycosylated, despite the presence of five potential N-glycosylation sites, and is rich in cysteine and hydrophobic amino acid residues. These features have suggested that LPHα folds rapidly into a tight and rigid globular domain in which carbohydrate attachment sites are no longer accessible to glycosyltransferases. In this paper we investigate the folding features of LPHβ and LPHβinitial and the putative role of LPHα as an intramolecular chaperone. Additionally, in view of the new constellation that the initial cleavage occurs at Arg734/Leu735 rather than at Arg868-Ala869, it became important to determine the role of the polypeptide stretch Leu735-Arg868in the processing and folding within the pro-LPH species.

      EXPERIMENTAL PROCEDURES

       Materials and Reagents

      Tissue culture dishes were obtained from Greiner, Hamburg, Germany. Streptomycin, penicillin, glutamine, Dulbecco's modified Eagle's medium (DMEM), methionine-free DMEM (denoted Met-free medium), and trypsin were purchased from Invitrogen, Eggenstein, Germany. Fetal calf serum, pepstatin, leupeptin, aprotinin, trypsin inhibitor, and molecular mass standards for SDS-PAGE were purchased from Sigma Chemical Co., Deisenhofen, Germany. Phenylmethylsulfonyl fluoride, antipain, and soybean trypsin inhibitor were obtained from Roche Diagnostics (Mannheim).l-[35S]Methionine (>1000 Ci/mmol) and protein A-Sepharose were obtained from Amersham Biosciences, Inc., Freiburg, Germany. Acrylamide,N,N′-methylenebisacrylamide, and TEMED were purchased from Carl Roth GmbH, Karlsruhe, Germany. SDS, ammonium persulfate, dithiothreitol, and Triton X-100 (TX-100) were obtained from Merck, Darmstadt/Germany. Endo-β-acetylglucosaminidase H (endo H) and endo-β-N-acetylglucosaminidase F/N-glycosidase F (endo F/GF) were purchased from New England BioLabs, Frankfurt, Germany. The pEYFP-N1 and pECFP-N1 vectors were purchased from CLONTECH Laboratories, Inc, Heidelberg, Germany. Restriction enzymes and T4-DNA-Ligase were obtained from MBI Fermentas, St. Leon-Rot, Germany.

       Immunochemical Reagents

      For immunoprecipitation of human LPH mouse monoclonal antibodies (mAb) of hybridoma HBB 1/909 (
      • Hauri H.P.
      • Sterchi E.E.
      • Bienz D.
      • Fransen J.A.
      • Marxer A.
      ) and MLac 2, MLac 6, and MLac 8 (
      • Maiuri L.
      • Raia V.
      • Potter J.
      • Swallow D.
      • Ho M.W.
      • Fiocca R.
      • Finzi G.
      • Cornaggia M.
      • Capella C.
      • Quaroni A.
      ) were used. The antibodies were generous gifts of Dr. Hans-Peter Hauri (Biozentrum, Basel, Switzerland), Dr. Erwin Sterchi (University of Bern, Switzerland), and Dr. Dallas Swallow (Medical Research Council, London, UK). LPHα was precipitated with the polyclonal antibody V496 directed against the N-terminal part of the prodomain (
      • Naim H.Y.
      • Jacob R.
      • Naim H.
      • Sambrook J.F.
      • Gething M.J.
      ). Anti-BiP and anti-calnexin antibodies were generous gifts from Dr. Neil Bulleid, University of Manchester, UK.

       Construction of cDNA Clones

      LPHβinitialwas cloned in two steps. First, the cDNA encoding the signal sequence of pro-LPH, LPHsignal, was amplified by PCR as described before (
      • Naim H.Y.
      • Jacob R.
      • Naim H.
      • Sambrook J.F.
      • Gething M.J.
      ). A second part of the cDNA comprising the last 20 nucleotides of the LPH signal sequence (nucleotides 52–71) and nucleotides 2203–3572 of LPHβinitial was synthesized by PCR using LPHcDNA as template and the oligonucleotides LPHsig/beta (GTT TTT CAT GCT GGG GGT CAC TGT TGC AGT TTG TAT CCC T) and cLPH3600 (
      • Naim H.Y.
      • Lacey S.W.
      • Sambrook J.F.
      • Gething M.J.
      ). Both DNA fragments were then fused by assembly PCR and cloned with the 3′-EcoRI/HindIII fragment of LPH cDNA into the unique EcoRI site of the pJB20 expression vector (
      • Jacob R.
      • Brewer C.
      • Fransen J.A.
      • Naim H.Y.
      ) by a three-way ligation. The resulting construct denoted pJB20-LPHβinitial was sequenced and found to contain the signal sequence of pro-LPH fused in-frame to the cDNA beginning with nucleotide 2203. For the generation of a CFP fusion protein the EcoRI/ScaI fragment of pJB20-LPHβinitial was subcloned into theEcoRI/SmaI-digested pECFP-N1 vector (CLONTECH, Inc., Heidelberg, Germany) to generate pLPHβinitial-CFP.
      The profragment of pro-LPH, LPHα, extending to nucleotide 2202 was first amplified from the LPHcDNA template by PCR with the primer pair LPH1 (
      • Naim H.Y.
      • Lacey S.W.
      • Sambrook J.F.
      • Gething M.J.
      )/cLPHα (AAT CTA GAG CCT CCT TAT CCC CCA G), which inserts a stop codon at position 2203 in the LPHαcDNA. This DNA fragment was inserted into the unique EcoRI/XbaI sites of pcDNA3 (Invitrogen, Groningen, The Netherlands). The resulting construct, pcDNA3-LPHα, was confirmed by sequencing. LPHα and YFP were fused by PCR amplification of LPHα using full-length LPHcDNA as template with the primer pair LPH1HindIII (AAA AGC TTC CTA GAA AAT GGA GCT G)/cLPHαSalI (CCT CGT CGA CCT CCT TAT CCC CCA GGG) and ligation of the PCR product into the HindIII/SalI sites of pEYFP-N1. The generated plasmid was confirmed by sequencing and named pLPHα-YFP.

       Transient Transfection of COS-1 Cells, Biosynthetic Labeling, and Immunoprecipitation

      COS-1 cells were transiently transfected with DNA by using DEAE-dextran essentially as described previously (
      • Naim H.Y.
      • Lacey S.W.
      • Sambrook J.F.
      • Gething M.J.
      ). pJB20-LPHβinitial, pcDNA3-LPHα, or a mixture of both plasmids, pLPHα-YFP and pLPHβinitial-CFP, were used. 48 h after transfection, the cells were biosynthetically labeled. Here, the cells were preincubated with 5 ml of methionine-free MEM for 1 h after which time the medium was changed and replaced by a similar medium containing 50 μCi of [35S]methionine. Labeling was performed for 1 h followed by a chase with non-labeled methionine for 4 h. After labeling, the cells were rinsed twice with cold phosphate-buffered saline and solubilized with 1 ml of lysis buffer containing 25 mm Tris-HCl (pH 8.0), 50 mm NaCl, 0.5% sodium deoxycholate, and 0.5% TX-100 supplemented with 1 mm phenylmethylsulfonyl fluoride, 1 μg/ml pepstatin, 5 μg/ml leupeptin, 5 μg/ml aprotinin, 1 μg/ml antipain, and 50 μg/ml trypsin inhibitor for 30 min at 4 °C. The cell extracts were centrifuged to remove cell debris while the supernatant was retained for the subsequent immunoprecipitation. After 1 h of preclearing with 20 μl of protein A-Sepharose, the immunoprecipitation was performed with polyclonal anti-LPHα (V496) or a mixture of mAb anti-LPH (MLac 2, MLac 6, and MLac 8) and 50 μl of protein A-Sepharose as described previously (
      • Jacob R.
      • Weiner J.R.
      • Stadge S.
      • Naim H.Y.
      ). For assessment of the interaction of the various LPHβinitial forms with BiP or calnexin, the cellular extracts were immunoprecipitated with mAb anti-BiP or mAb anti-calnexin (
      • Jacob R.
      • Bulleid N.J.
      • Naim H.Y.
      ). The immunoprecipitates were eluted by boiling in 1% SDS for 10 min followed by 10-fold dilution of SDS using a buffer containing 1% Triton X-100. Thereafter the samples were immunoprecipitated with a mixture of anti-LPH antibodies directed against native and denatured forms of the protein. The immunoprecipitates were further processed on SDS-PAGE.

       Trypsin Treatment of Cell Lysates

      LPHβinitialwas immunoprecipitated from transient transfected COS-1 cells labeled with [35S]methionine for 6 h. The immunoprecipitates were treated with 50 μg/ml trypsin for the indicated times at 37 °C. The reaction was stopped by the addition of 2 mg/ml soybean trypsin inhibitor (Roche Molecular Biochemicals, Mannheim, Germany).

       Cell-free Transcription/Translation

      The vector pcDNA3-LPHα containing the profragment of human pro-LPH was linearized with ApaI. Transcription was initiated with T7-RNA polymerase and carried out essentially as described before (
      • Jacob R.
      • Bulleid N.J.
      • Naim H.Y.
      ). 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. Translation of the newly synthesized RNA was performed in 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 ofl-[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 2 or 6 mm oxidized glutathione (GSSG) at 30 °C for 60 min. Microsomal membranes were isolated as described by Bulleid and Freedman (
      • Bulleid N.J.
      • Freedman R.B.
      ). Samples were prepared for electrophoresis by mixing with 5 vol. of SDS-PAGE sample buffer (62.5 mm Tris-HCl, pH 6.8, SDS (2% w/v), glycerol (10% w/v), and bromphenol blue) in the presence of 50 mmdithiothreitol for reducing conditions and for non-reducing conditions in the presence of 100 mm iodoacetamide and boiled for 5 min. After electrophoresis, gels were processed for autoradiography and exposed to Kodak X-Omat AR film.

       Confocal Fluorescence Microscopy

      Confocal images of living cells were acquired 2 days after transfection on a Leica TCS SP2 microscope with a ×63 water planapochromat lens (Leica Microsystems) (
      • Jacob R.
      • Naim H.Y.
      ). Dual color CFP and YFP images were obtained by sequential scans with the 458- and 514-nm excitation lines of an argon laser and the optimal emission wavelength for CFP or YFP, respectively.

       Enzymatic Assays

      Lactase activity was measured according to Dahlqvist (
      • Dahlqvist A.
      ) using lactose as substrate. LPHβinitialwas immunoprecipitated from cell lysates of transiently transfected COS-1 cells. For each determination of the lactase activity, the lysate equivalent to fourteen 100-mm-diameter dishes of confluent cells was utilized. The immunoprecipitates were subsequently assayed for lactase activity essentially as described (
      • Naim H.Y.
      • Lacey S.W.
      • Sambrook J.F.
      • Gething M.J.
      ).

       Other Procedures

      Digestion of 35S-labeled immunoprecipitates with endo-β-N-acetylglucosaminidase H (endo H) and endo-β-N-acetylglucosaminidase F/glycopeptidase F (endo F/GF) was performed as previously described (
      • Naim H.Y.
      • Sterchi E.E.
      • Lentze M.J.
      ). Neuraminidase treatment of immunoprecipitates was performed in 20 mm sodium citrate, 20 mm Tris-malate buffer (pH 6.0) and protease inhibitors for 2 h at 37 °C essentially as described by Jacob et al. (
      • Jacob R.
      • Brewer C.
      • Fransen J.A.
      • Naim H.Y.
      ).

      RESULTS

       The LPHβinitial Polypeptide Is a Temperature-sensitive Folding Mutant

      The cDNA encoding the initial cleavage product, LPHβinitial(Leu735-Phe1927) was fused to the signal sequence of pro-LPH to allow translocation across the ER membrane (Fig.1). Expression of this hybrid form in COS-1 cells and assessment of its glycosylation pattern using endo H sensitivity revealed a strongly labeled 150-kDa mannose-rich polypeptide (LPHβinitial/h) in addition to a 175-kDa endo H-resistant complex protein (LPHβinitial/c). The proportions of these two forms at steady state demonstrated a predominant presence of the mannose-rich protein with ∼70% of the total LPHβinitial protein. When LPHβfinalwas expressed individually the amount of complex-glycosylated LPHβfinal was substantially reduced (
      • Naim H.Y.
      • Jacob R.
      • Naim H.
      • Sambrook J.F.
      • Gething M.J.
      ) suggesting a role of the Leu735-Arg866 stretch in the processing of LPHβinitial. Nevertheless, the processing of mannose-rich LPHβinitial (LPHβinitial/h) to the complex-glycosylated form (LPHβinitial/c) was markedly less efficient in comparison with that of wild type pro-LPH (Fig. 2 A) pointing to an altered folding pattern of LPHβ in the absence of the profragment. The enzymatic activity of lactase has been assigned to residues Glu1273 and Glu1749 within the LPHβinitial domain (
      • Zecca L.
      • Mesonero J.E.
      • Stutz A.
      • Poiree J.C.
      • Giudicelli J.
      • Cursio R.
      • Gloor S.M.
      • Semenza G.
      ). Individual expression of this domain is associated with a drastic reduction in the lactase activity (Table I) reminiscent of a misfolded structure of LPHβinitial at least at or in the vicinity of the catalytic site. However, the partial conversion of LPHβinitial from a mannose-rich polypeptide into a complex-glycosylated protein suggests that a proportion of the LPHβinitial protein has acquired a correct conformation albeit at a slower rate. Alternatively, minimal folding requirements of LPHβinitial were fulfilled permitting the partially folded protein to egress the ER. We wanted therefore to determine whether slowing down the processing rate of LPHβinitialand prolonging its residence time in the ER influences the structural features of LPHβinitial and its biological activity. For this purpose, cells transfected with the cDNA encoding LPHβinitial were labeled biosynthetically at 20 °C. Fig. 2 B shows that the mannose-rich polypeptide was converted to a complex-glycosylated protein after 8 h of chase. Interestingly, this protein was now as enzymatically active as its wild type pro-LPH counterpart (Ref.
      • Naim H.Y.
      • Lacey S.W.
      • Sambrook J.F.
      • Gething M.J.
      and Table I). We next compared the fate of LPHβinitial under the two different labeling temperatures by employing a pulse-chase protocol. Here, a dramatic difference in the turnover rates was observed. Although LPHβinitial was gradually degraded with increasing chase periods at 37 °C, longer chase periods of up to 24 h at 20 °C have resulted in an efficient conversion of mannose-rich LPHβinitial/h to a meanwhile predominantly labeled complex-glycosylated LPHβinitial/c.
      Figure thumbnail gr1
      Figure 1Schematic representation of the structure of pro-LPH in human small intestinal cells and the two constructs LPH βinitial and LPH α. Some important structural features of pro-LPH in human small intestinal cells. Shown is the cleavable sequence (Met1-Gly19) at the N terminus. The ectodomain encompasses amino acid residues Ser20 to Thr1882. The intracellular proteolytic cleavage takes place between Arg734 and Leu735 to generate LPHβinitial. The luminal extracellular cleavage occurs at Arg868 to generate the brush-border mature enzyme, LPHβfinal (Arg868-Phe1927) (
      • Jacob R.
      • Radebach I.
      • Wuthrich M.
      • Grunberg J.
      • Sterchi E.E.
      • Naim H.Y.
      ,
      • Wuthrich M.
      • Grunberg J.
      • Hahn D.
      • Jacob R.
      • Radebach I.
      • Naim H.Y.
      • Sterchi E.E.
      ). The construct LPHβinitial was generated by fusing the 19 amino acids of the LPH signal sequence to Leu735, the N terminus of LPHβinitial. For expression of LPHα, a stop codon was introduced after Arg734. MA, membrane anchoring; CT, cytoplasmic tail.
      Figure thumbnail gr2
      Figure 2Biosynthesis and transport kinetics of LPH βinitial in COS-1 cells. A, COS-1 cells were transiently transfected with pJB20-LPHβinitial, biosynthetically labeled for 6 h followed by immunoprecipitation of LPHβinitial. The immunoprecipitates were divided into three aliquots and treated with endo H, endo F/GF, or not treated. The proteins were subjected to SDS-PAGE and autoradiography. B, transiently transfected COS-1 cells were pulsed with [35S]methionine for 1 h at 37 °C followed by different chase times at 20 °C or 37 °C. Cells were lysed, and the immunoprecipitates were analyzed by SDS-PAGE and autoradiography. C, immunoprecipitates of transiently transfected COS-1 cells incubated at 20 °C or 37 °C were loaded onto SDS-PAGE followed by Coomassie Blue staining of the gel.
      Table IEnzymatic activity of LPHβinitial in transfected COS cells
      TemperatureLPHβinitialLPHβinitial + LPHα
      IU/nmol
      20 °C2.6 ± 0.4ND
      ND, not determined.
      37 °C0.2 ± 0.11.2 ± 0.4
      COS-1 cells were transiently transfected with pJB20-LPHβinitial alone or in combination with pcDNA3-LPHα and incubated at 37 °C for 48 h. Following cell lysis LPHβinitial was immunoprecipitated and the lactase activities in the immunoprecipitates were measured according to Dahlqvist (
      • Dahlqvist A.
      ). Results are the mean ± S.E. of three experiments.
      1-a ND, not determined.
      This increase in stability of LPHβinitial after synthesis at 20 °C was also observed by determination of the steady-state level of the polypeptide after synthesis at 37 °C or 20 °C in a Coomassie Blue-stained polyacrylamide gel (Fig. 2 C). In these experiments LPHβinitial/h was isolated from transfected COS cells incubated at 37 °C, whereas LPHβinitial/c was detected in cells cultured at 20 °C. This indicates that reduced temperatures facilitate the formation of a stable conformation of complex-glycosylated LPHβinitial/c. A direct comparison of the forms generated at the two different temperatures reveals an increase of ∼10 kDa in the apparent molecular mass of LPHβinitial/cformed at 37 °C as compared with the isoform at 20 °C (Fig.3 A). One possible explanation for this difference could be altered sugar contents in both forms. To determine this content, pulse-chase experiments, with transfected cells at 37 °C or 20 °C combined with deglycoslyation of the LPHβinitial isoforms with neuraminidase and endo F/GF, were performed (Fig. 3 A). At 4 h of chase the mannose-rich glycosylated LPHβinitial/h was immunoprecipitated from the cell lysates, which was sensitive to endo F/GF and expectedly devoid of sialic acid as assessed by its insensitivity to neuraminidase treatment. Within 12 h of chase complex-glycosylated LPHβinitial/c appeared and shifted to 155 kDa after desialylation with neuraminidase. Neuraminidase also reduces LPHβinitial/c synthesized at 37 °C to the 155-kDa polypeptide. Therefore, the difference in the apparent molecular masses of the LPHβinitial at both temperatures is due to a variable content of terminal sialic acid residues. To analyze if this difference in glycosylation of LPHβinitial/c has any influence on or is related to changes in the folding and stability of the protein, we probed its sensitivity toward trypsin. Correctly folded wild type brush-border LPHβ is resistant to trypsin treatment (
      • Naim H.Y.
      • Lacey S.W.
      • Sambrook J.F.
      • Gething M.J.
      ). Changes in the protein folding of LPHβinitial would be therefore monitored by variation in its susceptibility to trypsin. As shown in Fig.3 B, trypsin resulted in a drastic degradation of LPHβinitial/h and LPHβinitial/c, which were synthesized at 37 °C. After 5 min of incubation with trypsin, almost all of the LPHβinitial was degraded. By contrast, LPHβinitial synthesized and processed at 20 °C was resistant to trypsin. It is worthwhile to note that a minor proportion of LPHβinitial synthesized at 37 °C was resistant to trypsin, and, interestingly enough, this proportion had a similar apparent molecular mass as the correctly folded LPHβinitial synthesized at 20 °C. The acquisition of trypsin resistance at 20 °C similar to the wild type enzyme strongly suggests that LPHβinitial exhibits characteristics of a temperature-sensitive protein in the absence of the LPHα profragment. The variation in the folding pattern observed at 37 °C is also associated with an altered terminal glycosylation pattern.
      Figure thumbnail gr3
      Figure 3Processing and folding of LPH βinitial at 20 °C. A, COS-1 cells were transiently transfected with pJB20-LPHβinitial, biosynthetically labeled at 20 °C for 4 h, and chased for the indicated times or labeled at 37 °C with [35S]methionine for 2 h followed by a 12-h chase. After cell lysis LPHβinitial was immunoprecipitated and the immunoprecipitates were divided into equal aliquots and treated with endo F/GF, neuraminidase, or not treated. The proteins were subjected to SDS-PAGE and autoradiography. B, trypsin sensitivity assay of LPHβinitial. Transiently transfected COS-1 cells were biosynthetically labeled at 37 °C for 8 h or at 20 °C for 12 h followed by immunoprecipitation of LPHβinitial from the cell lysates. The immunoprecipitates were treated with trypsin for different times and analyzed on SDS-PAGE. The trypsin-sensitive form of LPHβinitial/c detected after synthesis at 37 °C is indicated by asterisks.

       LPHα Alone Is a Compact Globular Polypeptide

      Obviously the presence of the LPHα profragment is crucial in influencing the folding of LPHαinitial in the context of wild type pro-LPH. It has been previously postulated that the LPHα molecule, which contains 11 cysteine residues, attains a compact structure that is stabilized by disulfide bridges and that acts as a kernel for the folding of the mature enzyme (
      • Naim H.Y.
      • Jacob R.
      • Naim H.
      • Sambrook J.F.
      • Gething M.J.
      ). To characterize the role of this domain in this context, we assessed its structural features in anin vitro translation assay. The cDNA encoding LPHα was transcribed in vitro and translated with rabbit reticulocyte lysate supplemented with dog pancreatic microsomal vesicles. This system has already been used to demonstrate the presence of disulfide bridges in the full-length pro-LPH enzyme (
      • Jacob R.
      • Bulleid N.J.
      • Naim H.Y.
      ). The main polypeptide synthesized in the absence of added microsomal vesicles had an apparent molecular mass of 90 kDa (Fig.4 A) consistent with the molecular weight of LPHα 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 100 kDa was generated. Separation of the microsomal membranes from the reticulocyte lysates by centrifugation through a sucrose cushion revealed a predominant labeling of this species in the microsomal membranes. The 100-kDa protein is mannose-rich and glycosylated, because it was sensitive to treatment with endo H and converted to the nascent unglycosylated 90-kDa polypeptide. The nascent 90-kDa translation product was therefore exposed to the glycosylation machinery in the lumen of the microsomal membranes. To investigate the presence of cotranslationally formed disulfide bonds, the translation reaction was carried out in the presence of oxidized glutathione (GSSG). GSSG oxidizes existing thiol groups and facilitates, therefore, the formation of disulfide bonds. It is required in the reaction mixture to compensate for the presence of the reducing agent dithiothreitol in the commercially prepared reticulocyte lysates. 2 or 6 mm GSSG was 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.
      ). In the absence of GSSG and under non-reducing conditions, both the 90-kDa as well as the glycosylated 100-kDa polypeptides could be detected (Fig.4 B). On the other hand, the addition of GSSG to the translation mixture results in a substantial increase in the mobility of the microsomal form of LPHα from a 100-kDa species to one of ∼70 kDa under non-reducing conditions. The faster migration behavior of LPHα through the gel matrix is reminiscent of a compact conformation of this form facilitated by the formation of intramolecular disulfide bonds in the presence of GSSG. The primary sequence of LPHα contains 11 cysteine residues as putative candidates for the formation of disulfide bridges, and the obvious mobility shift observed implies that more than one disulfide bond might be formed to reach that high degree of condensation.
      Figure thumbnail gr4
      Figure 4Cell-free synthesis of LPH α. A, LPHα was synthesized in the presence or absence of microsomal membranes (MM) and in the presence of varying concentrations of the oxidant GSSG as indicated (B). The microsomal membranes were isolated by centrifugation through a sucrose cushion and treated with endo H or not treated. The samples were finally analyzed by SDS-PAGE on 8% slab gels followed by autoradiography. For non-reducing conditions (B) the samples were treated with 100 mm iodoacetamide and loaded on 8% SDS-PAGE. A compact form of LPHα that migrates faster through the gel is indicated by asterisks.

       LPHα Resides in the ER of Transfected COS-1 Cells

      We further analyzed the structural features of LPHα in vivoafter expression in COS-1 cells. LPHα was immunoprecipitated from biosynthetically labeled transfected cells with the polyclonal antibody V496, which binds to the N-terminal part of LPHα (
      • Naim H.Y.
      • Jacob R.
      • Naim H.
      • Sambrook J.F.
      • Gething M.J.
      ), followed by treatment of the immunoprecipitates with endo H or endo F/GF. Fig.5 A shows that three forms of LPHα were revealed. A major 100-kDa component similar to theN-glycosylated polypeptide was synthesized in the cell-free system and two minor 103- and 97-kDa polypeptides. All three forms were converted to the non-glycosylated 90-kDa species uponN-deglycosylation with either endo H or endo F/GF, indicating that they correspond to various glycosylated isoforms of the same polypeptide. Furthermore, by virtue of the specificity of endo H in cleaving mannose-rich N-glycans and endo F/GF in cleaving mannose-rich as well as complex N-linked glycans, the data indicate that LPHα is exclusively mannose-rich and glycosylated and is consequently located in the ER and is not further transported to the Golgi apparatus.
      Figure thumbnail gr5
      Figure 5LPH α is blocked in the ER. A, COS-1 cells were transiently transfected with pcDNA3-LPHα and labeled with [35S]methionine for 6 h. Following cell lysis, LPHα was immunoprecipitated with pAb V496. The immunoprecipitates were further treated with endo H, endo F/GF, or not treated and analyzed by SDS-PAGE and autoradiography.B, for confocal studies COS-1 cells were transiently transfected with pLPHα-YFP followed by 48-h incubation at 37 °C. A confocal analysis of the cells was performed in a Leica TCS-SP2 microscope, which indicated a strong ER-specific staining. Scale bar, 20 μm.
      These findings were corroborated by confocal microscopic analyses of COS-1 cells expressing the yellow fluorescence protein (YFP) fused to LPHα (LPHα-YFP). The transfected cells revealed exclusive labeling of the ER (Fig. 5 B).

       LPHβinitial Polypeptide Acquires Enzymatic Activity and Trypsin-resistant Conformation in the Presence of LPHα

      Having assessed the structural features of LPHβinitial and LPHα individually we wanted next to examine directly the role of LPHα on the folding of LPHβinitial. One approach is to examine whether the enzymatic activity of LPHβinitial increases in the presence of LPHα reminiscent of acquisition of correct folding. For this, COS-1 cells were cotransfected with the cDNAs encoding LPHβinitial and LPHα. In the control cells LPHβinitial was individually expressed. As shown above, the enzymatic activity of LPHβinitial expressed alone in cells cultured at 37 °C was very low, whereas this protein was enzymatically active only at the permissive temperature of 20 °C (Table I). On the other hand, the cotransfected cells reveal a dramatic elevation in the enzymatic activity of LPHβinitial.
      In the second approach the conformation of LPHβinitial in the presence or absence of LPHα was probed with trypsin to determine whether alterations in the folding pattern have occurred. LPHβinitial was expressed in COS cells alone or in the presence of LPHα. The biosynthetically labeled proteins were immunoprecipitated with mAb anti-LPH and treated with trypsin or with V496, the polyclonal anti-LPHα antibody. Fig.6 shows that in the absence of cotransfected LPHα almost half of the LPHβinitial was cleaved by trypsin to a smaller polypeptide pair corresponding to the mannose-rich and complex-glycosylated forms. Immunoprecipitation of the cellular lysates with V469, the anti-LPHα antibody, was utilized as an internal control to confirm the absence of this protein in this experimental set up. On the other hand, coexpression of LPHβinitial with LPHα leads to a substantial decrease in the proportion of LPHβinitial cleaved by trypsin to less than 10% reminiscent of an increased stability of this protein. The presence of the LPHα protein was confirmed by it reactivity with the V496 antibody. It is established that the 160-kDa wild type brush-border LPHβ is trypsin-resistant (
      • Naim H.Y.
      • Lacey S.W.
      • Sambrook J.F.
      • Gething M.J.
      ). That a complete resistance of LPHβinitial to trypsin has not been achieved is most likely due to the heterogeneous transfected cell populations, which contained both genes, or the individual ones, and therefore an optimal effect on LPHβinitial could be attained. This is also the reason why the enzymatic activity could not be fully restored to normal brush-border levels.
      Figure thumbnail gr6
      Figure 6Trypsin treatment of LPH βinitial isolated from COS-1 cells. COS-1 cells were transiently transfected with pJB20-LPHβinitial in the presence or absence of pcDNA3-LPHα. 48 h after transfection the cells were labeled with [35S]methionine for 8 h. The immunoprecipitated LPHβinitial was treated with trypsin for 10 min at 37 °C. The reaction was stopped by the addition of soybean trypsin inhibitor followed by boiling in SDS-PAGE sample buffer. The samples were finally analyzed by SDS-PAGE on 6% slab gels and autoradiography.
      Finally, a significantly higher proportion of the complex-glycosylated species in the total synthesized LPHβinitial protein was detected in presence of LPHα as compared with that of the individually expressed LPHβinitial (69.8%versus 79.2%). This increase suggests a more efficient processing and transport rate of mannose-rich LPHβinitialas a consequence of altered folding characteristics in the presence of LPHα.
      In conclusion, LPHβinitial is a temperature-sensitive protein that is characterized by an improperly folded, trypsin-sensitive, and biologically inactive protein at 37 °C. Normal structural and functional features could be restored either at 20 °C or by addition of the prodomain LPHα, indicating that these N-terminal 734 amino acids of the proregion would assist the folding of LPHβinitial at 37 °C.

       LPHα Is Associated with LPHβinitial When Expressed in COS-1 Cells

      Having assessed the role of LPHα on LPHβinitial, we wanted to determine how this effect ensues and whether there is a direct interaction between the two proteins in the ER, where the most crucial folding events take place. For this, LPHβinitial and LPHα were coexpressed in COS-1 cells and each protein was immunoprecipitated from the detergent extracts of biosynthetically labeled cells, either with mAb anti-LPH antibody, directed against LPHβinitial, or pAb V496 antibody directed against the LPHα. Fig.7 A demonstrates that both species coimmunoprecipitated with either antibody despite the specific affinity of the antibodies used toward a particular epitope found on the individual subunits and not on both. The question that arises is whether the association between both subunits of pro-LPH results in the attainment of a native conformation of LPHβinitial. This is indeed the case, because there is an increase in the proportion of LPHβinitial/c due to the presence of LPHα intrans and its direct association after biosynthesis with LPHβinitial. The low amount of LPHα coprecipitating with LPHβ is compatible with a transient interaction occurring between these two forms that is terminated as soon as the LPHβ protein has acquired a correct conformation. Moreover, this interaction appears to be specific to LPH, because the LPHα prosequence is not capable of modulating the folding of the isomaltase or sucrase subunits of the sucrase-isomaltase enzyme complex.
      R. Jacob, B. Pürschel, and H. Y. Naim, manuscript in preparation..
      Neither the isomaltase nor the sucrase subunits are correctly folded species when they are individually expressed, and they require each other to be transported along the secretory pathway. Here the presence of the LPHα could not substitute for the role of either subunit in the folding process of the other.
      Figure thumbnail gr7
      Figure 7Association of LPH α and LPH βinitial in the ER elevates the proportion of complex-glycosylated LPH βinitial. A, coprecipitation of LPHα and LPHβinitial. COS-1 cells were transiently transfected with pcDNA3-LPHα and pJB20-LPHβinitial and labeled 48 h post transfection with [35S]methionine for 8 h. The cell lysates were immunoprecipitated with mAb directed against the mature LPHβ molecule. As a control, the supernatants were subsequently immunoprecipitated with V496 directed against the LPHα-propeptide. Immunoprecipitated samples were further analyzed on SDS-PAGE and autoradiography. B, confocal analysis of LPHβinitial-CFP and LPHα-YFP. COS-1 cells were transfected with pLPHβinitial-CFP (blue) in the absence (a) or presence of LPHα-YFP (yellow) (b–d). Expression of LPHβinitial-CFP alone (a) results in an ER-specific staining. After cotransfection with pLPHα-YFP, the LPHβinitial-CFP molecules (b) were further transported to the Golgi apparatus with arrows indicating transport vesicles. LPHα-YFP is shown in c, and anoverlay of b and c is presented ind (LPHα-YFP in yellow, LPHβinitial-CFP in blue). (Scale bars: a, 10 μm; b–d, 20 μm.) Thearrows show transport vesicles containing LPHβinitial-CFP when expressed in the presence of LPHα-YFP.
      To visualize the effect of LPHα on LPHβ in life cells, the two proteins were fused either to the cyan fluorescent protein, CFP (LPHβinitial-CFP), or the yellow fluorescent protein, YFP (LPHα-YFP). Confocal microscope imaging of transfected COS-1 cells revealed a predominant ER localization of the LPHβinitial-CFP (Fig. 7 B, panel a). On the other hand, when both subunits, LPHα-YFP and LPHβinitial-CFP, were coexpressed in the same cell, LPHβinitial-CFP could be localized in the Golgi apparatus, in transport vesicles and also at the cell surface (Fig.6 B, panels b–d). Here, both species were expressed at virtually similar levels as assessed by quantification of the specific fluorescence, excluding an imbalanced expression of the subunits. In the absence of LPHα-YFP no significant labeling of LPHβinitial-CFP in the Golgi or at the cell surface could be observed (Fig. 7 B, panel a). Together, these observations support the biochemical data, which showed that the individual expression of LPHβ results in a smaller proportion of mature and complex-glycosylated LPHβ as compared with wild type pro-LPH and LPHβ coexpressed with LPHα (see TableII, Fig. 6, and Ref.
      • Naim H.Y.
      • Jacob R.
      • Naim H.
      • Sambrook J.F.
      • Gething M.J.
      ).
      Table IIProcessing of LPHβinitial to a complex glycosylated protein increases in the presence of LPHα
      LPHβinitialLPHβinitial + LPHα
      %
      Mannose-rich glycosylated30.2 ± 3.620.8 ± 3.2
      Complex glycosylated69.8 ± 3.679.2 ± 3.2
      COS-1 cells were transiently transfected with pJB20-LPHβinitial alone or in combination with pcDNA3-LPHα and incubated at 37 °C for 48 h. After pulse labeling for 1 h with [35S]methionine and a chase of 4 h, LPHβinitial was immunoprecipitated and analyzed by SDS-PAGE on 6% slab gels and fluorography. Results of densitometric scanning are the mean ± S.E. of five experiments.

       The ER-resident Molecular Chaperones BiP and Calnexin Assist the Folding of LPHβ

      The trans effect of LPHα on the folding of LPHβinitial has lead us to ask how this effect would associate and conform with other folding events that implicate ER components, such as BiP and calnexin (
      • High S.
      • Lecomte F.J.
      • Russell S.J.
      • Abell B.M.
      • Oliver J.D.
      ). The putative and temporal interaction of LPHβinitial with BiP and calnexin was analyzed in transiently transfected COS-1 cells either expressing LPHβinitial alone or in addition of LPHα. A pulse-chase analysis of these cells followed by coprecipitation of chaperone-bound LPHβinitial with anti-BiP and anti-calnexin antibodies is demonstrated in Fig. 8. After a pulse period of 10 min the mannose-rich polypeptide of individually expressed LPHβinitial (i.e. in the absence of LPHα) could be found associated with BiP, but not calnexin. Essentially a reversed binding pattern was obtained within 30 min of chase. Here, LPHβinitial bound to calnexin and only slightly to BiP. The same pattern was also revealed after 60 min of chase. Later, at 120 min of chase, however, the earliest binding profiles of LPHβinitial to the two chaperones were repeated,i.e. strong binding to BiP and almost negligible or no binding to calnexin. Obviously LPHβinitial is subject to several cycles of association, dissociation, and reassociation with ER-resident proteins. Each of these cycles commences with the binding of BiP to an early folding intermediate of LPHβinitialfollowed by its dissociation and the association with calnexin, which in turn dissociates followed by the reassociation of BiP. The prolonged period of binding to these two chaperones to LPHβinitialat virtually similar intensities throughout is presumably due to an inefficient folding of LPHβinitial.
      Figure thumbnail gr8
      Figure 8Sequential interaction of LPH βinitial with BiP and calnexin. COS-1 cells were transfected with a combination of pcDNA3-LPHα and pJB20-LPHβinitial or pJB20-LPHβinitial. 48 h post transfection, the cells were labeled for 10 min followed by different chase intervals. The cell lysates were immunoprecipitated with mAb anti-LPH and when indicated with anti-BiP or anti-calnexin. The samples were analyzed on SDS-PAGE and autoradiography.
      The sequential pattern of association of LPHβinitial with BiP and calnexin changes substantially when LPHβinitialwas coexpressed with LPHα. Here, only BiP, but not calnexin, bound to LPHβinitial. The labeling intensity of LPHβinitial that was associated with BiP decreased concomitantly with the appearance of complex-glycosylated forms (Fig.8). In sum, the data demonstrate that the absence of the LPHα prodomain exposes binding sites for calnexin resulting in the retention of partially folded forms of LPHβinitial by this chaperone and a repeated association with BiP. LPHα, on the other hand, assists the folding of LPHβinitial to a transport-competent form that leaves the ER for further transport to the cell surface. At least in part, calnexin compensates for the absence of LPHα by retaining LPHβinitial until BiP binds and the protein goes into a new folding cycle.

      DISCUSSION

      The initial intracellular proteolytic cleavage of intestinal pro-LPH in the trans-Golgi network at Arg734/Leu735 (
      • Jacob R.
      • Radebach I.
      • Wuthrich M.
      • Grunberg J.
      • Sterchi E.E.
      • Naim H.Y.
      ) is a major processing event along the secretory pathway of this protein. The generated LPHβinitial is sorted to the apical membrane, where it undergoes another cleavage step by trypsin to generate the brush-border LPH protein, LPHβfinal (
      • Jacob R.
      • Radebach I.
      • Wuthrich M.
      • Grunberg J.
      • Sterchi E.E.
      • Naim H.Y.
      ,
      • Wuthrich M.
      • Grunberg J.
      • Hahn D.
      • Jacob R.
      • Radebach I.
      • Naim H.Y.
      • Sterchi E.E.
      ). Two independent studies have previously provided strong evidence that individual expression of sequences encoding brush-border LPH, i.e.Arg867-Phe1927, result in a transport-incompetent and enzymatically inactive protein (
      • Oberholzer T.
      • Mantei N.
      • Semenza G.
      ,
      • Naim H.Y.
      • Jacob R.
      • Naim H.
      • Sambrook J.F.
      • Gething M.J.
      ) thus implicating the profragment in the folding events of pro-LPH as an intramolecular chaperone.
      In the present report we addressed the putative role of LPHα as an intramolecular chaperone as well as the interplay between this domain, the LPHβinitial, and two ER-resident proteins. These data demonstrate that LPHβinitial is a temperature-sensitive protein that folds properly to an active protein at 20 °C but is misfolded and inactive under the physiological temperature. The first folding steps of proteins destined for transport along the secretory pathway occur in the ER, which harbors a folding machinery comprising a battery of molecular chaperones. Two members of this machinery, BiP and calnexin, interact with LPHβinitial at the non-permissive temperature, whereby this interaction follows a sequential pattern and most likely proceeds for several cycles. The cycles of association, dissociation, and reassociation with the chaperone and the longer residence of the LPHβinitial protein in the ER are apparently not sufficient for the attainment of the protein to a mature, transport-competent configuration. In fact, only a minor proportion of this protein egresses the ER and acquires a complex type of glycosylation in the Golgi. Despite traversing the quality control machinery in the ER, these polypeptides are enzymatically inactive and sensitive toward trypsin treatment. This is reminiscent of altered folding that is also reflected by a variation in theN-glycosylation pattern, particularly that of the sialic acid content, as compared with the correctly folded protein. A dual function of molecular chaperones in regulating the folding of proteins has been described for many proteins. In contrast to LPHβinitial, however, the proteins in these cases acquire ultimately a correctly folded configuration. For example, the folding of the G protein of the vesicular stomatitis virus is critically dependent on the interaction of this protein with BiP and calnexin (
      • Hammond C.
      • Helenius A.
      ), whereas the immunoglobulin chains interact with BiP and GRP94 (
      • Melnick J.
      • Dul J.L.
      • Argon Y.
      ). The assembly of the multitransmembrane domains of the cystic fibrosis transmembrane conductance regulator (CFTR) protein in the cytosol is facilitated through the sequential binding with the chaperones hdj-2 and hsc-70 (
      • Meacham G.C.
      • Lu Z.
      • King S.
      • Sorscher E.
      • Tousson A.
      • Cyr D.M.
      ), and in the case of thyroglobulin the interaction with molecular chaperones follows a precursor-product relationship (
      • Kim P.S.
      • Arvan P.
      ).
      The defect in the folding pattern of LPHβinitial at the non-permissive temperature can be only overcome when the N-terminally located LPHα is present. Here, enzymatic activity as well as correct folding is restored. One of the early examples of prodomains with a folding function as an intramolecular chaperone is the prodomain of subtilisin (,
      • Inouye M.
      ). After maturation of the precursor molecule, this polypeptide is proteolytically cleaved to generate the biologically active protease. It can guide the folding of the inactive protein to an active proteasein vitro when added exogenously (
      • Zhu X.L.
      • Ohta Y.
      • Jordan F.
      • Inouye M.
      ). Intramolecular chaperone function has also been described for the prodomains of activin A (
      • Gray A.M.
      • Mason A.J.
      ), transforming growth factor β1 (
      • Gray A.M.
      • Mason A.J.
      ), cathepsin C (
      • Cigic B.
      • Dahl S.W.
      • Pain R.H.
      ), and type 1 matrix metalloproteinase (
      • Cao J.
      • Hymowitz M.
      • Conner C.
      • Bahou W.F.
      • Zucker S.
      ). The 13.5-kDa N-terminal part of cathepsin C folds spontaneously and rapidly to a compact monomer with stable tertiary interactions (
      • Cigic B.
      • Dahl S.W.
      • Pain R.H.
      ). It is involved in the sequential formation of disulfide linkages in the native protein. This has also been observed for the 13-residue proregion of bovine pancreatic trypsin inhibitor, which provides an intramolecular thiol disulfide reagent capable of assisting and accelerating disulfide bond formation in the native enzyme (
      • Weissman J.S.
      • Kim P.S.
      ). In an oxidizing milieu such as that of the ER the LPHα domain assumes a compact configuration stabilized by disulfide bridges that link several of the 11 cysteine residues (see “Results”)
      R. Jacob, K. Peters, and H. Y. Naim, unpublished observations.
      and facilitated by hydrophobic interaction of almost 45% of uncharged, non-polar amino acid residues that are likely to be oriented into the interior space of this protein. These structural features are favored by the electrophoretic behavior of the faster migrating oxidized LPHα on SDS gels as compared with the non-oxidized isoform as well as the reduced N-glycosylation. Potential N-linked glycosylation sites in proteins are usually more accessible to glycosylation in unfolded proteins than their folded and compact counterparts (
      • Allen S.
      • Naim H.Y.
      • Bulleid N.J.
      ). In analogy with other profragments and by virtue of its decisive function in the folding of LPHβinitial one would assume that LPHα facilitates the accurate formation of disulfide bridges in the LPHβinitial domain, which are indispensable for the correct folding and generation of a native pro-LPH (
      • Jacob R.
      • Bulleid N.J.
      • Naim H.Y.
      ). Moreover, the direct binding of LPHα to LPHβinitial, as shown in the in vitro assays, suggests that LPHα could function to prevent misfolding, to permit an autonomous folding of LPHβinitial, and to help generate disulfide bonds in the ER. It should be noted that the interaction of the LPHα prosequence is most likely specific to LPH, because it is not capable of modulating the folding of the isomaltase subunit of the sucrase-isomaltase enzyme complex. Here, neither the isomaltase nor the sucrase subunits are competently transported along the secretory pathway when they are individually expressed.
      By virtue of the striking sequence similarities between LPHα and LPHβinitial it is likely that LPHα builds a kernel structure immediately after synthesis that functions as a folding template for other homologous structural domains of LPHβinitial. This would also explain why several cycles of sequential interaction of LPHβinitial with the two chaperones, BiP and calnexin, in the absence of LPHα are not sufficient for LPHβinitial to acquire correctly folded confirmation whereas the binding of LPHα was.
      Current concepts suggest that at least two additional mechanisms specify the function of intramolecular chaperones, an unfolding of misfolded conformations, and a nonspecific prevention of protein misfolding. The mode of action of LPHα on the folding of LPHβinitial provides an additional novel mechanism of this type of domains.
      The breakage of the cycles of sequential binding of LPHβinitial to BiP and calnexin, in the presence of LPHα, prevent the interaction of LPHβinitial with calnexin, suggesting that both polypeptides, LPHα and calnexin, compete in binding to common sites on LPHβinitial. Nevertheless, calnexin by itself is not able to replace LPHα, because it does not share homologies with LPHβinitial as LPHα does and is therefore unable to behave as a folding template for LPHβinitial.

      Acknowledgments

      We thank Dr. Neil Bulleid, University of Manchester, United Kingdom, Dr. Hans-Peter Hauri, Biozentrum, University of Basel, Dr. Erwin Sterchi, Institute of Biochemistry and Molecular Biology, University of Bern, Switzerland, and Dr. Dallas Swallow, Medical Research Council, London, United Kingdom for generous gifts of monoclonal anti-BiP, anti-calnexin, and anti-LPH antibodies.

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