Additional N-Glycosylation and Its Impact on the Folding of Intestinal Lactase-phlorizin Hydrolase

Lactase-phlorizin hydrolase (LPH) is a membrane bound intestinal hydrolase, with an extracellular domain comprising 4 homologous regions. LPH is synthesized as a large polypeptide precursor, pro-LPH, that undergoes several intra- and extracellular proteolytic steps to generate the final brush-border membrane form LPHβfinal. Pro-LPH is associated through homologous domain IV with the membrane through a transmembrane domain. A truncation of 236 amino acids at the COOH terminus of domain IV (denoted LAC236) does not significantly influence the transport competence of the generated mutant LPH1646MACT (Panzer, P., Preuss, U., Joberty, G., and Naim, H. Y. (1998) J. Biol. Chem. 273, 13861–13869), strongly suggesting that LAC236 is an autonomously folded domain that links the ectodomain with the transmembrane region. Here, we examine this hypothesis by engineering several N-linked glycosylation sites into LAC236. Transient expression of the cDNA constructs in COS-1 cells confirm glycosylation of the introduced sites. The N-glycosyl pro-LPH mutants are transported to the Golgi apparatus at substantially reduced rates as compared with wild-type pro-LPH. Alterations in LAC236 appear to sterically hinder the generation of stable dimeric trypsin-resistant pro-LPH forms. Individual expression of chimeras containing LAC236, the transmembrane domain and cytoplasmic tail of pro-LPH and GFP as a reporter gene (denoted LAC236-GFP) lends strong support to this view: while LAC236-GFP is capable of forming dimersper se, its N-glycosyl variants are not. The data strongly suggest that the LAC236 is implicated in the dimerization process of pro-LPH, most likely by nucleating the association of the ectodomains of the enzyme. Lactase-phlorizin hydrolase (LPH) is a membrane bound intestinal hydrolase, with an extracellular domain comprising 4 homologous regions. LPH is synthesized as a large polypeptide precursor, pro-LPH, that undergoes several intra- and extracellular proteolytic steps to generate the final brush-border membrane form LPHβfinal. Pro-LPH is associated through homologous domain IV with the membrane through a transmembrane domain. A truncation of 236 amino acids at the COOH terminus of domain IV (denoted LAC236) does not significantly influence the transport competence of the generated mutant LPH1646MACT (Panzer, P., Preuss, U., Joberty, G., and Naim, H. Y. (1998) J. Biol. Chem. 273, 13861–13869), strongly suggesting that LAC236 is an autonomously folded domain that links the ectodomain with the transmembrane region. Here, we examine this hypothesis by engineering several N-linked glycosylation sites into LAC236. Transient expression of the cDNA constructs in COS-1 cells confirm glycosylation of the introduced sites. The N-glycosyl pro-LPH mutants are transported to the Golgi apparatus at substantially reduced rates as compared with wild-type pro-LPH. Alterations in LAC236 appear to sterically hinder the generation of stable dimeric trypsin-resistant pro-LPH forms. Individual expression of chimeras containing LAC236, the transmembrane domain and cytoplasmic tail of pro-LPH and GFP as a reporter gene (denoted LAC236-GFP) lends strong support to this view: while LAC236-GFP is capable of forming dimersper se, its N-glycosyl variants are not. The data strongly suggest that the LAC236 is implicated in the dimerization process of pro-LPH, most likely by nucleating the association of the ectodomains of the enzyme. endoplasmic reticulum lactase-phlorizin hydrolase (all forms) uncleaved precursor of LPH monoclonal antibody mannose-rich precursor complex glycosylated precursor a stretch of 236 amino acids (Arg1647-Thr1882) located juxtapose the membrane anchoring domain of pro-LPH green fluorescent protein polyacrylamide gel electrophoresis endoglycosidase N,N,N′,N′-tetramethylethylenediamine The pathways by which membrane and secretory proteins attain their three-dimensional structure, in particular the implication of glycosylation in these events has been the target of extensive investigation in the past few years. N-Glycosylation is necessary for efficient folding in the ER1 (1.Fan H. Meng W. Kilian C. Grams S. Reutter W. Eur. J. Biochem. 1997; 246: 243-251Crossref PubMed Scopus (75) Google Scholar, 2.Letourneur O. Sechi S. Willette-Brown J. Robertson M.W. Kinet J.P. J. Biol. Chem. 1995; 270: 8249-8256Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar, 3.Riederer M.A. Hinnen A. J. Bacteriol. 1991; 173: 3539-3546Crossref PubMed Google Scholar, 4.Zhang Y. Dahms N.M. Biochem. J. 1993; 295: 841-848Crossref PubMed Scopus (27) Google Scholar, 5.Roberts P.C. Garten W. Klenk H.D. J. Virol. 1993; 67: 3048-3060Crossref PubMed Google Scholar), interaction with calnexin and calreticulin (6.Cannon K.S. Hebert D.N. Helenius A. J. Biol. Chem. 1996; 271: 14280-14284Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar), receptor-ligand binding (7.Zhang R. Cai H. Fatima N. Buczko E. Dufau M.L. J. Biol. Chem. 1995; 270: 21722-21728Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar), transport to lysosomes (8.Chapman R.L. Kane S.E. Erickson A.H. J. Biol. Chem. 1997; 272: 8808-8816Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar), polarized sorting to the apical plasma membrane (9.Scheiffele P. Peranen J. Simons K. Nature. 1995; 378: 96-98Crossref PubMed Scopus (417) Google Scholar), and also for optimal expression of some proteins (10.Shen F. Wang H. Zheng X. Ratnam M. Biochem. J. 1997; 327: 759-764Crossref PubMed Scopus (37) Google Scholar). The addition ofN-linked core oligosaccharides to membrane and secretory glycoproteins occurs co-translationally at asparagine residues in the tripeptide sequon Asn-Xaa-Ser/Thr soon after translocation of the nascent polypeptide into the lumen of the endoplasmic reticulum (ER). However, the presence of the sequon does not ensure core glycosylation, as many proteins contain sequons that remain either unglycosylated or glycosylated to a variable extent (11.Allen S. Naim H.Y. Bulleid N.J. J. Biol. Chem. 1995; 270: 4797-4804Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar). N-Glycosyl sugar chains are core-glycosylated in the ER and become complex glycosylated by passing through the Golgi apparatus. AlthoughN-glycosylation in the ER constitutes the critical step in the initial folding of proteins, the complex glycosylated chains in some glycoproteins, acquired in the Golgi, are also required for the acquisition of a correct conformation (12.Loo T.W. Clarke D.M. J. Biol. Chem. 1998; 273: 14671-14674Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar). Several roles forN-linked oligosaccharides in protein folding have been described so far. Their presence assists folding by facilitating disulfide bond formation (11.Allen S. Naim H.Y. Bulleid N.J. J. Biol. Chem. 1995; 270: 4797-4804Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar, 13.Feng W. Matzuk M.M. Mountjoy K. Bedows E. Ruddon R.W. Boime I. J. Biol. Chem. 1995; 270: 11851-11859Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar, 14.Rickert K.W. Imperiali B. Chem. Biol. 1995; 2: 751-759Abstract Full Text PDF PubMed Scopus (55) Google Scholar) or through a chaperone-mediated glucose trimming and reglycosylation cycle in the ER (15.Hebert D.N. Foellmer B. Helenius A. Cell. 1995; 81: 425-433Abstract Full Text PDF PubMed Scopus (490) Google Scholar). The absence of N-glycosyl chains influences the quaternary structure of proteins as has been demonstrated for the dimerization of MUC2 mucin (16.Asker N. Axelsson M.A.B. Olofsson S.-O. Hansson G.C. J. Biol. Chem. 1998; 273: 18857-18863Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar). Also highly glycosylated protein domains, like the carboxyl-terminal peptide (CTP) of the human placental hormone CG-β subunit, can participate in the folding of the whole subunit (17.Muyan M. Boime I. Mol. Endocrinol. 1998; 12: 766-772PubMed Google Scholar). We used human lactase-phlorizin hydrolase (EC 3.2.1.23–3.2.1.62) (LPH) as the appropriate model protein to study the influence ofN-glycosyl sugar chains on the folding of single protein domains. This enzyme is synthesized in intestinal cells as a pro-LPH that undergoes one intracellular proteolytic cleavage in the Golgi apparatus to generate a mature LPHβinitial that is targeted with high fidelity to the apical membrane. A final cleavage step takes place in the intestinal lumen by trypsin to generate LPHβfinal that exerts its biological function in hydrolyzing lactose. Pro-LPH consists of four homologous regions with 38–55% homology and is associated with the plasma-membrane via a membrane anchor at the COOH terminus of the LPHβfinaldomain (18.Mantei N. Villa M. Enzler T. Wacker H. Boll W. James P. Hunziker W. Semenza G. EMBO J. PubMed Scopus Google Scholar). The protein are likely or in the folding of pro-LPH. is the critical of the in the of LPHβinitial M. J. R. I. Naim H.Y. Biochem. 1996; PubMed Scopus Google Scholar, R. I. M. J. Naim H.Y. Eur. J. Biochem. 1996; PubMed Scopus Google Scholar). of this domain to a in the of folded and H.Y. R. Naim H. J. Biol. Chem. Full Text PDF PubMed Google T. N. Semenza G. 1993; PubMed Scopus (27) Google Scholar). the the transmembrane and also the cytoplasmic of pro-LPH a in acquisition of a correct dimeric protein structure H.Y. Naim H. Eur. J. Biol. 1996; Google Scholar) and transport competence of the enzyme P. G. Naim H.Y. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar). The homologous IV a with of regions in a of membrane proteins as and W. M. Semenza G. Cell. Full Text PDF PubMed Scopus Google Scholar, J. E. J. J. Hunziker W. PubMed Scopus Google Scholar). This domain the of the We that of 236 amino acids located in the of the membrane in homologous IV influence on the quaternary structure, and sorting of LPH to the plasma membrane P. G. Naim H.Y. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar). a of amino acids of the stretch have to an of the dimerization and to a in the transport of the This that the 236 amino acids of domain IV of the extracellular domain and function as a the protein and the membrane by forming a region. of the amino acids of the an structure to the of O. H. PubMed Scopus Google Scholar). to data a of large of sites Y. G. Biochem. J. 1991; PubMed Scopus Google Scholar), the amino acids of the contain several which are glycosylated in the mature form of LPH acids H.Y. J. Biol. Chem. Full Text PDF PubMed Google Scholar). this we the of this domain in the of the folding of pro-LPH by sites into this domain. We that the constructs are at and transported to the at rates the wild-type The of the sites is in to the folding of a the 236 amino acids the dimerization and of the and and for and protein and and and of and with the in The used in this for the for the for the The containing the cDNA of LPH into the of H.Y. J. Biol. Chem. 1991; Full Text PDF PubMed Google Scholar) used for of the of LPH into the This the for with the in to confirm the and into the LPH expression in COS-1 the and expression by the LPH cDNA into the of the H.Y. J. Biol. Chem. 1991; Full Text PDF PubMed Google Scholar) containing LPH into the of to the cDNA for a pro-LPH with GFP the generation of the LAC236-GFP the of synthesized at by LPH cDNA as as described H.Y. R. Naim H. J. Biol. Chem. Full Text PDF PubMed Google Scholar). cDNA comprising the of and to of pro-LPH synthesized by or as and the and The to in four The and into the of to and The of the constructs by in with and (denoted or of the appropriate as described H.Y. J. Biol. Chem. 1991; Full Text PDF PubMed Google Scholar). COS-1 cells with of in containing and (denoted for at by a with for of The cells with with of as described P. G. Naim H.Y. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar). The to and the with lactase-phlorizin hydrolase monoclonal antibody A. J. Biol. PubMed Scopus Google Scholar) or with the antibody and with protein the protein with A in and with in to of the by and with constructs containing GFP also with and the proteins by to A. Biochem. PubMed Scopus Google Scholar) as of COS-1 cells of used for of The for as described by Naim H.Y. J. Biol. Chem. 1991; Full Text PDF PubMed Google Scholar). COS-1 cells with of the trypsin at for or and the by the addition of of trypsin at 4 by in to P. G. Naim H.Y. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar, COS-1 cells for with with and in of and a of and the for at at 4 The a to or to for at at 4 and with or and on of with and as described H.Y. Biochem. J. PubMed Scopus Google Scholar). of 236 amino acids of the membrane anchor of LPH not the quaternary structure, transport or polarized sorting of the enzyme P. G. Naim H.Y. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar). data that this stretch which in the homologous IV of is folded and is a the transmembrane of LPH and the of its this hypothesis and and the of this the ectodomain of we introduced that in sites The mutants one of the amino to to and to The influence of these on the and transport of pro-LPH in cells with expression containing the LPH an of the of mutant of these with demonstrated the of glycosylation of these forms. The to their complex of glycosylation, while the and to with wild-type pro-LPH, these to the mannose-rich and complex glycosylated and by with The introduced sites in and demonstrated to glycosylated compared with wild-type pro-LPH. The mannose-rich as as their complex glycosylated in their as compared with their wild-type by the to the complex glycosylated the mannose-rich of the mutants and wild-type pro-LPH to in the of the complex glycosylated pro-LPH wild-type pro-LPH of the proteins the mannose-rich pro-LPH are of the an in is the of at the sites. addition to in the of the complex glycosylated of the mutants as compared with wild-type pro-LPH We compared the transport of and with of wild-type pro-LPH by a The are in and wild-type pro-LPH as mannose-rich after of pro-LPH in the Golgi apparatus the of complex glycosylated pro-LPH while 4 to required to the complex glycosylated of and of wild-type pro-LPH to a complex glycosylated of of of and of as complex glycosylated an the that the glycosylation mutants transported to the Golgi at a wild-type of wild-type pro-LPH and in COS-1 COS-1 cells for or and with with the into and with or not The by on of wild-type pro-LPH and mutant COS-1 cells with the cDNA of wild-type pro-LPH and the for with and for the of with The by on of the in The reduced transport rates of the pro-LPH glycosylation mutants is most likely the of an three-dimensional structure as has been for (1.Fan H. Meng W. Kilian C. Grams S. Reutter W. Eur. J. Biochem. 1997; 246: 243-251Crossref PubMed Scopus (75) Google Scholar, 2.Letourneur O. Sechi S. Willette-Brown J. Robertson M.W. Kinet J.P. J. Biol. Chem. 1995; 270: 8249-8256Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar, 3.Riederer M.A. Hinnen A. J. Bacteriol. 1991; 173: 3539-3546Crossref PubMed Google Scholar, 4.Zhang Y. Dahms N.M. Biochem. J. 1993; 295: 841-848Crossref PubMed Scopus (27) Google Scholar, 5.Roberts P.C. Garten W. Klenk H.D. J. Virol. 1993; 67: 3048-3060Crossref PubMed Google Scholar). We the folding of the mutants as compared with wild-type pro-LPH by and COS-1 cells with the of and wild-type pro-LPH and for with The with of by to confirm the expression of the while the for as a The the to The of the mutants that the mutants are not The wild-type pro-LPH as with a to that H.Y. J. Biol. Chem. 1991; Full Text PDF PubMed Google Scholar). The of pro-LPH has been to residues and in LPH H. P. R. G. Semenza G. J. Biol. Chem. Full Text PDF PubMed Google Scholar). for the of in the mutants are which in a to the in and We the folding of the mutants by a trypsin folded pro-LPH to a LPHβfinal mature enzyme which is H.Y. J. Biol. Chem. 1991; Full Text PDF PubMed Google Scholar). A in the trypsin cleavage is with protein trypsin cleavage sites become which are in the core of the folded protein H.Y. J. Biol. Chem. 1991; Full Text PDF PubMed Google R. Bulleid N.J. Naim H.Y. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar). Pro-LPH COS-1 cells with for and the with trypsin for or at in 4 the LPHβfinal of trypsin and at the and after of a not appear the mutants with to the pro-LPH in the these of trypsin the of and the to by trypsin strongly suggest that and have protein folding as compared with pro-LPH. The acquisition of pro-LPH to transport competence occurs in the ER and dimerization of the mannose-rich This the membrane anchoring domain of pro-LPH, the of which in an of of pro-LPH in the ER and an A folded ectodomain is not for dimerization to H.Y. Naim H. Eur. J. Biol. 1996; Google Scholar). The in the of complex glycosylated of the mutants to the synthesized and is of a transport of the We to examine in the quaternary structure of the mutants have also this the dimerization of the mutants in The are in data (18.Mantei N. Villa M. Enzler T. Wacker H. Boll W. James P. Hunziker W. Semenza G. EMBO J. PubMed Scopus Google Scholar, H.Y. Naim H. Eur. J. Biol. 1996; Google Scholar), mannose-rich wild-type pro-LPH in the in and the dimeric in the The complex glycosylated dimeric wild-type pro-LPH in the the mutants mannose-rich pro-LPH in the (7.Zhang R. Cai H. Fatima N. Buczko E. Dufau M.L. J. Biol. Chem. 1995; 270: 21722-21728Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar, R.L. Kane S.E. Erickson A.H. J. Biol. Chem. 1997; 272: 8808-8816Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar, P. Peranen J. Simons K. Nature. 1995; 378: 96-98Crossref PubMed Scopus (417) Google Scholar) as as the dimeric in the is that the mutants to acquisition of complex of as has been demonstrated for wild-type pro-LPH. However, the in the of the dimeric of the mutants to wild-type pro-LPH as as the of these in a of dimerization of the mutants as compared with wild-type pro-LPH. We that the reduced dimerization of the mutants is for their of to complex glycosylated forms. the membrane anchoring domain and the have not been in the mutants and the are to in the extracellular of pro-LPH, we that in the ectodomain of pro-LPH have or the of dimerization of the of the that N-glycosyl mutants in the LAC236 domain are associated with reduced dimerization and transport of pro-LPH a of the LAC236 domain in these events a of LAC236 in the dimerization we this domain in with the transmembrane domain of pro-LPH in Also the N-glycosyl variants described that are this domain to the of for the LAC236 domain we used GFP as a reporter gene that also to the of these the The GFP protein to the of a comprising LAC236 or its the transmembrane and the cytoplasmic tail with these constructs for with and the with The wild-type LAC236-GFP a of a polypeptide and a these to one single that the are glycosylation The of the protein to that for the LAC236-GFP The of the that this co-translationally mannose-rich glycosylated in the not in the Golgi apparatus to a mature complex glycosylated a to the and also as mannose-rich the as by their The of these to the sites. of the chimeras by the GFP the in of an ER and the for the not support the that the chimeras are proteins and not the the GFP to wild-type pro-LPH not its transport competence as demonstrated by the acquisition of the to a complex glycosylated and This is by the in which at the in the Golgi apparatus and in on the transport of pro-LPH is that GFP have been the of of the LAC236-GFP and in COS-1 COS-1 cells with expression or The cells with and with a we the quaternary structure of the and its N-glycosyl variants by in of the that at in the to a form of the while the the a dimeric form of as by protein on is that the protein mannose-rich The with wild-type pro-LPH and its variants that in the LAC236 domain the dimerization of pro-LPH. that is a dimeric we to the of the sites on the dimerization of this that the extent of dimerization of LAC236 mutants has been the of the mutants in the containing many that of wild-type LAC236 with reduced dimerization of the the of N-glycosyl sites has the dimerization The data support the that LAC236 is critical in the dimerization of pro-LPH and in this as by are to the dimerization of the is that dimerization of LAC236 not for this form to the ER or to the Golgi apparatus. The acquisition of a protein to its final a of complex events in the ER Helenius A. Biol. PubMed Scopus Google R.W. Helenius A. 1993; PubMed Scopus Google Scholar) in some in the Golgi apparatus O. A. M. R. A. Cell. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). and protein folding by interaction of residues in the polypeptide formation of as and and formation of disulfide or that particular regions of the protein a J. Nature. PubMed Scopus Google Scholar). The folding of a large as pro-LPH, is to a complex process that and the of three-dimensional of pro-LPH is on the of the four homologous regions (18.Mantei N. Villa M. Enzler T. Wacker H. Boll W. James P. Hunziker W. Semenza G. EMBO J. PubMed Scopus Google Scholar) or the W. P. N. J. 1991; Google Scholar) in the LPH However, and of mutants of LPH have some on the of polypeptide in the intracellular and The large for exerts its function as an and is in the correct folding of the located This of pro-LPH H.Y. Naim H. Eur. J. Biol. 1996; Google Scholar, J. Biochem. 1995; PubMed Scopus Google Scholar), an that the transmembrane domain and is required for of LPH the ER H.Y. Naim H. Eur. J. Biol. 1996; Google Scholar). The of the investigation is a that is located of the membrane in the homologous IV and 236 amino acids of this pro-LPH influence on the quaternary structure and folding of the mutant as as on its transport competence and polarized sorting P. G. Naim H.Y. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar) that this autonomously and is not a of critical that are implicated in events of Here, we examine this by several N-linked glycosylation sites to regions which are to form or The of N-linked glycosylation sites these of the structure of the However, the addition of a as an that an to amino acids and to the The addition of oligosaccharides can used to particular regions on the of pro-LPH that are and that are in protein transport and of the mutants generated has one This is to substantially many of the and of pro-LPH. of the mutants is as transport as wild-type the mutants complex of and have been transported through the Golgi the of the mature form pro-LPH is substantially reduced as compared with the wild-type This is the of a in the dimerization of the mutants and dimerization is required for pro-LPH to transport competence and the ER H.Y. Naim H. Eur. J. Biol. 1996; Google Scholar, J. Biochem. 1995; PubMed Scopus Google Scholar). is that engineering an in LAC236 on the transport of pro-LPH while of the does not significantly influence the and sorting of the pro-LPH P. G. Naim H.Y. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar). to the LAC236 stretch in the of the pro-LPH Individual expression of the LAC236 domain to the transmembrane domain of pro-LPH and the reporter gene GFP that this domain is capable of This is reduced or N-glycosyl sites are the that as a mannose-rich polypeptide is of transport this dimerization is not a for a protein to the ER and of LAC236 the ER of the pro-LPH has been that of the LAC236 domain a LPH1646MACT mutant P. G. Naim H.Y. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar). However, the in the transport of this mutant the ER to the Golgi apparatus to the pro-LPH to a of LAC236 in the initial of of the ectodomain of pro-LPH. in the of LAC236 to are not to the domain have on the folding of the of pro-LPH, the P. G. Naim H.Y. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar). This is in in which N-glycosyl mutants are by trypsin in to wild-type pro-LPH of in the structure of pro-LPH Individual expression of LAC236 and its N-glycosyl mutants that the N-glycosyl chains sterically hinder the association of LAC236 of one with that of a is that these in the folding of the not dimerization of the the generated are not stable trypsin of a association the folding of the LAC236 domain are required in the of pro-LPH, in this domain by the of the pro-LPH LAC236 the association of the ectodomain of pro-LPH and a of this is necessary and for this process to of the of LAC236, is on some of this domain. The LAC236 stretch is in amino acids (18.Mantei N. Villa M. Enzler T. Wacker H. Boll W. James P. Hunziker W. Semenza G. EMBO J. PubMed Scopus Google Scholar) and that is of The sites in the wild-type as as the introduced are in the the of pro-LPH is located at in this is to folding in the mutants by the of pro-LPH. the mutants are is a of in the folding of the domain. to these the the amino have the folding of a in a to the This is likely for the mutant in which one of residues has been by a to a The mutants and with located the not this The is that in LAC236 to of this region. This in this domain a critical in the or the folding and the transport of pro-LPH. Pro-LPH is to with H.Y. J. Biol. Chem. Full Text PDF PubMed Google Scholar), which has been to place in the domain. A also forN-linked Several sites are in this domain (18.Mantei N. Villa M. Enzler T. Wacker H. Boll W. James P. Hunziker W. Semenza G. EMBO J. PubMed Scopus Google Scholar) and is that not of these sites are with the data one that addition of to in the folding of pro-LPH. this model the LAC236 domain a in the of structure and function of pro-LPH. We of and of and of for of the monoclonal