Site-directed mutagenesis of the human thyrotropin receptor: role of asparagine-linked oligosaccharides in the expression of a functional receptor

We studied the role of glycosylation in the expression of a functional human TSH receptor. Oligonucleotide-directed mutagenesis was used to replace, separately or together, the Asn codons with Gln in each of the six potential glycosylation sites in the receptor. Recombinant wild-type and mutated TSH receptors were stably expressed in Chinese hamster ovary cells. High affinity TSH binding and the cAMP response to TSH stimulation were abolished in the receptor mutated at Asn77 as well as in the receptor mutated at all six potential glycosylation sites. In the receptor mutated at Asn113, the affinity of TSH binding was markedly decreased (Kd, 2.6 x 10(-8) 3.3 x 10(-10) M in the wild-type receptor). This affinity was too low to permit the transduction of a signal, as measured by an increase in intracellular cAMP generation. Substitution of Asn at positions 99, 177, 198, and 302 did not appreciably affect the affinity of the TSH receptor for TSH binding or its ability to mediate an increase in intracellular cAMP levels. Therefore, either these four potential glycosylation sites are not glycolysated, or alternatively, oligosaccharide chains at these positions do not play a major role in the folding, intracellular trafficking, stability, or expression of a functional receptor on the cell surface. Conversely, our data suggest that N-linked glycosylation of Asn77 and Asn113 does play a role in the expression of a biologically active TSH receptor on the cell surface.     

Functional role of N-linked glycosylation on the rat melanin-concentrating hormone receptor 1

Melanin-concentrating hormone (MCH) is known to act through two G-protein-coupled receptors MCHR1 and MCHR2. MCHR1 has three potential sites (Asn13, Asn16 and Asn23) for N-linked glycosylation in its extracellular amino-terminus which may modulate its reactivity. Site-directed mutagenesis of the rat MCHR1 cDNA at single or multiple combinations of the three potential glycosylation sites was used to examine the role of the putative carbohydrate chains on receptor activity. It was found that all three potential N-linked glycosylation sites in MCHR1 were glycosylated, and that N-linked glycosylation of Asn23 was necessary for full activity. Furthermore, disruption of all three glycosylation sites impaired proper expression at the cell surface and receptor activity. These data outline the importance of the N-linked glycosylation of the MCHR1.     

Posttranslational modifications of the lutropin receptor: mass spectrometric analysis

Our present knowledge of the lutropin (LH/hCG) receptor structure derives from deductions made from its amino acid sequence as established by studying the cDNA. To obtain direct experimental information, luteinizing hormone (LH) receptor expressed in L cells was immunopurified in sufficient amounts to warrant analysis by mass spectrometry and microsequencing. The mature receptor, complexed to human chorionic gonadotropin (hCG), was purified by using monoclonal antibodies recognizing the hormone, whereas the mannose-rich non-hormone-binding precursor was purified by use of antireceptor antibodies. Determination of the N-terminus showed that (2)/(3) of protein molecules started at Thr24 whereas (1)/(3) started at Ala28. All these molecules bound hCG, suggesting that the most N-terminal region of the receptor does not participate in hormone binding. Six N-glycosylation sites have been predicted from the amino acid sequence. One of them (Asn299) was found to be nonglycosylated in both the precursor and the mature protein. The most heavily glycosylated residue was Asn291, followed by Asn195 and Asn99. These three sites accounted for 82% and 97% of carbohydrate moieties in the mature receptor and in the mannose-rich precursor, respectively. The presence of some receptor molecules nonglycosylated at sites 99, 174, and 195 in hormone-receptor complexes dismisses a direct role of these glycosylation sites in hormone binding or in the correct folding of the protein. The mature carbohydrate chains were homogeneous at position 174, 195, and 313 (absence of Golgi mannosidase II activity at positions 174 and 313, absence of GlcNAc tranferases III and IV activity at position 195). Heterologous carbohydrates were present at sites 99 and 291. The latter, which is highly variable in carbohydrate chains, is unlikely to participate in a direct interaction with hormone. Site 313 thus remains as the main candidate for a role in hormone binding.     

Role of asparagine-linked oligosaccharides in the function of the rat PTH/PTHrP receptor

The receptor for parathyroid hormone (PTH) and PTH-related peptide (PTHrP) is a G-protein-coupled receptor with four potential sites for N-linked glycosylation. The contribution of the oligosaccharide moieties to cell surface expression, ligand binding, and signal transduction was investigated. Site-directed mutagenesis of the rat PTH/PTHrP receptor cDNA was performed at single or combination of the four potential glycosylation sites to determine the effect of the putative carbohydrate chains on the activities of the receptor. The results revealed that all four potential N-glycosylation sites in the PTH/PTHrP receptor are glycosylated. Receptors missing a single or multiple glycosylation consensus but with at least one intact glycosylation site expressed sufficiently and functioned normally. In contrast, the nonglycosylated receptor, in which all four glycosylation sites were mutated, is deficient in these functions. These data indicate important roles for N-linked glycosylation in PTH/PTHrP receptor functions.     

Identification of the N-linked glycosylation sites on the high density lipoprotein (HDL) receptor SR-BI and assessment of their effects on HDL binding and selective lipid uptake

The murine class B, type I scavenger receptor mSR-BI, a high density lipoprotein (HDL) receptor that mediates selective uptake of HDL lipids, contains 11 potential N-linked glycosylation sites and unknown numbers of both endoglycosidase H-sensitive and -resistant oligosaccharides. We have examined the consequences of mutating each of these sites (Asn --> Gln or Thr --> Ala) on post-translational processing of mSR-BI, cell surface expression, and HDL binding and lipid transport activities. All 11 sites were glycosylated; however, disruption of only two (Asn-108 and Asn-173) substantially altered expression and function. There was very little detectable post-translational processing of these two mutants to endoglycosidase H resistance and very low cell surface expression, suggesting that oligosaccharide modification at these sites apparently plays an important role in endoplasmic reticulum folding and/or intracellular transport. Strikingly, although the low levels of the 108 and 173 mutants that were expressed on the cell surface exhibited a marked reduction in their ability to transfer lipids from HDL to cells, they nevertheless bound nearly normal amounts of HDL. Indeed, the affinity of (125)I-HDL binding to the 173 mutant was similar to that of the wild-type receptor. Thus, N-linked glycosylation can influence both the intracellular transport and lipid-transporter activity of SR-BI. The ability to uncouple the HDL binding and lipid transport activities of mSR-BI by in vitro mutagenesis should provide a powerful tool for further analysis of the mechanism of SR-BI-mediated selective lipid uptake.     

Mutagenesis and gene transfer define site-specific roles of the gonadotropin oligosaccharides

Human chorionic gonadotropin (hCG), luteinizing hormone (LH), follicle-stimulating hormone and thyroid-stimulating hormone are a family of glycoprotein hormones that share a common alpha subunit but differ in their hormone-specific beta subunits. Using site-directed mutagenesis and gene-transfer, we analyzed the role of the N-linked oligosaccharides of alpha and chorionic gonadotropin (CG)beta in the secretion, assembly, and biologic activity of hCG. Absence of carbohydrate at alpha asparagine (Asn) 52 decreased combination with CG beta but did not alter monomer secretion. Absence of the alpha Asn78 oligosaccharide increased the degradation of the alpha subunit, but the presence of CG beta stabilized this alpha mutant in an efficiently formed dimer complex. Alternatively, absence of both alpha oligosaccharides slowed both secretion and dimer formation but allowed an intermediate level of alpha secreted or dimerized compared to the single-site mutants. Analysis of the CG beta glycosylation mutants revealed that absence of the Asn30 oligosaccharide, but not Asn13, slowed secretion but not assembly, whereas absence of both oligosaccharides slowed both secretion and dimer formation. Analysis of the receptor binding of the hCG glycosylation mutants showed that absence of any or all of the hCG N-linked oligosaccharides had only a minor effect on receptor affinity of the derivatives. However, the absence of alpha Asn52, but not the alpha Asn78 or the CG beta carbohydrate units, reduced the steroidogenic effect, unmasked differences in the beta oligosaccharides, and converted the deglycosylated derivatives into antagonists.     

Structural analysis and localization of the carbohydrate moieties of a soluble human interferon gamma receptor produced in baculovirus-infected insect cells.

A soluble form of the human interferon gamma receptor that is required for the identification of interferon gamma antagonists was expressed in baculovirus-infected insect cells. The protein carried N-linked carbohydrate and showed a heterogeneity on denaturing polyacrylamide gels. We investigated the utilization of the potential sites for N-linked glycosylation and the structure of the carbohydrate moieties of this soluble receptor. Amino acid sequence analysis and ion spray mass spectrometry revealed that of the five potential sites for N-linked glycosylation, Asn17 and Asn69 were always utilized, whereas Asn62 and Asn162 were utilized in approximately one-third of the protein population. Asn223 was never found to be glycosylated. The soluble receptor was treated with N-glycosidase F and the oligosaccharides released were analyzed by matrix-assisted laser desorption mass spectrometry, which showed that the protein carried six types of short carbohydrate chains. The predominant species was a hexasaccharide of molecular mass 1,039, containing a fucose subunit linked to the proximal N-acetylglucosamine residue: [formula: see text]     

N-linked glycosylation of G. mellonella juvenile hormone binding protein - comparison of recombinant mutants expressed in P. pastoris cells with native protein

Juvenile hormone (JH) regulates insect growth and development. JH present in the hemolymph is bound to juvenile hormone binding protein (hJHBP) which protects JH from degradation. In G. mellonella, this protein is glycosylated only at one (Asn(94)) of the two potential N-linked glycosylation sites (Asn(4) and Asn(94)). To investigate the function of glycosylation, each of the two potential glycosylation sites in the rJHBP molecule was examined by site-directed mutagenesis. MS analysis revealed that rJHBP overexpressed in the P. pastoris system may appear in a non-glycosylated as well as in a glycosylated form at both sites. We found that mutation at position Asn(94) reduces the level of protein secretion whereas mutation at the Asn(4) site has no effect on protein secretion. Purified rJHBP and its mutated forms (N4W and N94A) have the same JH binding activities similar to that of hJHBP. However, both mutants devoid of the carbohydrate chain are more susceptible to thermal inactivation. It is concluded that glycosylation of JHBP molecule is important for its thermal stability and secretion although it is not required for JH binding activity.     

Glycosylation affects agonist binding and signal transduction of the rat somatostatin receptor subtype 3.

The somatostatin receptor subtypes, sst1-sst5, bind their natural ligands, somatostatin-14, somatostatin-28 and cortistatin-17, with high affinity but do not much discriminate between them. Detailed understanding of the interactions between these receptors and their peptide ligands may facilitate the development of selective compounds which are needed to identify the biological functions of individual receptor subtypes. The influence of the amino-terminal domain and of the two putative N-linked glycosylation sites located in this region of rat sst3 was analysed. Biochemical studies in transfected cell lines suggested that the amino-terminus of sst3 is glycosylated at both sites. Mutation of the N-linked glycosylation site, Asn18Thr, had only a small effect on binding properties and inhibition of adenylyl cyclase. The double mutant Asn18Thr/Asn31Thr lacking both glycosylation sites showed a significant reduction in high affinity binding and inhibition of adenylyl cyclase while peptide selectivity was not affected. Truncation of the amino-terminal region by 32 amino acid residues including the two glycosylation sites caused similar but much stronger effects. Immunocytochemical analysis of receptor localisation revealed that the amino-terminal domain but not the carbohydrates appear to be involved in the transport of the receptor polypeptide to the cell surface.     

Glycosylation sites in the atrial natriuretic peptide receptor: oligosaccharide structures are not required for hormone binding.

Atrial natriuretic peptide (ANP) is a hormone involved in cardiovascular homeostasis through its natriuretic and vasodilator actions. The ANP receptor that mediates these actions is a glycosylated transmembrane protein coupled to guanylate cyclase. The role of glycosylation in receptor signaling remains unresolved. In this study, we determined, by a combination of HPLC/MS and Edman sequencing, the glycosylation sites in the extracellular domain of ANP receptor (NPR-ECD) from rat expressed in COS-1 cells. HPLC/MS analysis of a tryptic digest of NPR-ECD identified five glycosylated peptide fragments, which were then sequenced by Edman degradation to determine the glycosylation sites. The data revealed Asn-linked glycosylation at five of six potential sites. The type of oligosaccharide structure attached at each site was deduced from the observed masses of the glycosylated peptides as follows: Asn13 (high-mannose), Asn180 (complex), Asn306 (complex), Asn347 (complex), and Asn395 (high-mannose and hybrid types). Glycosylation at Asn180 and Asn347 was partial. The role of glycosyl moieties in ANP binding was examined by enzymatic deglycosylation of NPR-ECD followed by binding assay. NPR-ECD deglycosylated with endoglycosidase F2 and endoglycosidase H retained ANP-binding activity and showed an affinity for ANP similar to that of untreated NPR-ECD. Endoglycosidase treatment of the full-length ANP receptor expressed in COS-1 cells also had no detectable effect on ANP binding. These results suggest that, although glycosylation may be required for folding and transport of the newly synthesized ANP receptor to the cell surface, the oligosaccharide moieties themselves are not involved in hormone binding.     

Glycosylation at specific sites of erythropoietin is essential for biosynthesis, secretion, and biological function

The glycoprotein hormone erythropoietin (Ep), the primary regulator of erythropoiesis, is synthesized by the kidney and secreted as the mature protein with three N-linked and one O-linked oligosaccharide chains. To investigate the role(s) of each carbohydrate moiety in the biosynthesis and function of Ep, we have used oligonucleotide-directed mutagenesis of a cDNA for human Ep to alter the amino acids at each of the carbohydrate attachment sites. Each mutated cDNA construct was expressed in stably transfected sublines of a kidney cell line, baby hamster kidney. We show, by preventing attachment of N-linked carbohydrate at asparagines 38 or 83, or preventing O-linked glycosylation at serine 126, that glycosylation of each of these specific sites is critical for proper biosynthesis and secretion of Ep. Fractionation of cellular extracts demonstrated that the mutant proteins lacking glycosylation at each of these three sites, (38, 83, and 126) were associated mainly with membrane components or were degraded rapidly. Less than 10% of these three mutant proteins were processed properly and secreted from the cells. The Ep protein lacking N-linked glycosylation at asparagine 24 is synthesized and secreted as efficiently as native Ep. The carbohydrates at positions 24 and 38 may be involved in the biological activity of Ep, since the absence of either of the oligosaccharide side chains at these positions reduced the hormone's biological activity.     

The effect of losing glycosylation sites near the receptor-binding region on the receptor phenotype of the human influenza virus H1N1

The receptor properties of influenza virus (IF) isolates/SSSR/90/77 are studied. The isolates are peculiar for losing glycosylation sites (GS) at the Asn131 receptor-binding region (GS131) after passaging in mice and at the Asn158 region (GS158) after cultivation in the presence of mouse serum. The loss of each carbohydrate residue increases the influenza virus affinity for carbohydrate chains with the terminal group Neu5Ac alpha 2-6Gal and reduces its affinity for Neu5Ac alpha 2-3Gal receptors. The effect is more pronounced in the GS158-depleted virus. Upon substitution of asparagine by aspartic acid, the electrostatic component of virus binding to the receptor is altered because of the increased negative charge on hemagglutinin. The virus receptor phenotype changes depending on the cultivation conditions. The isolate adapted to mice has higher affinity to mouse lung cell receptors, while the virus propagated in chick embryos in the presence of inhibitors has higher affinity to allantoic membrane cells.     

Preparation of Recombinant Carbohydrate Deficient Active Analogs of Human Chorionic Gonadotropin from Insect Cells

Abstract

Human chorionic gonadotropin (hCG) has four N-glycosyl chains, two in each subunit. Several analogs lacking one or more specific N-linked carbohydrate chains have been purified from insect cells by immunoaffinity chromatography on a monoclonal antibody, B17, column Traces of the hCGβ mutant present, if any, were removed by a second immunoaffinity chromatography on a column of hCGβ specific monoclonal antibody, B158. N-glycosylation was inhibited by the replacement of either Asn or Thr to Gln in the consensus sequence, -Asn × Ser/Thr-, for N-glycosylation. All analogs were overexpressed in High-Five insect cells with the expression levels ranging between 1.5 to 15 μg/ml and were found homogeneous by SDS-PAGE under nonreducing and reducing conditions. Their molecular sizes ranged between 34k to 44k. The receptor binding affinity of all the analogs was unaltered as determined by radio receptor assay using rat ovarian membranes. The availability of these analogs should facilitate studies on the effect of a specific carbohydrate chain on the conformation and in vivo properties of hCG.     

Localization of N-linked carbohydrate chains in glycoprotein ZPA of the bovine egg zona pellucida.

The zona pellucida, a transparent envelope surrounding the mammalian oocyte, consists of three glycoproteins, ZPA, ZPB and ZPC, and plays a role in sperm-egg interactions. In bovines, these glycoproteins cannot be separated unless the acidic N-acetyllactosamine regions of the carbohydrate chains are removed by endo-beta-Galactosidase digestion. Endo-beta-Galactosidase-digested ZPB retains stronger sperm-binding activity than ZPC. It is still unclear whether ZPA possesses significant activity. Recently, we reported that bovine sperm binds to Man5GlcNAc2, the neutral N-linked chain in the cow zona proteins. In this study, we investigated the localization of the sperm-ligand active high-mannose-type chain and the acidic complex-type chains in bovine ZPA. Three N-glycopeptides of ZPA, containing an N-glycosylation site at Asn83, Asn191 and Asn527, respectively, were obtained from endo-beta-Galactosidase-digested ZPA. Of these glycosylation sites, only Asn527 is present in the ZP domain common to all the zona proteins. The carbohydrate structures of the N-linked chains obtained from each N-glycopeptide were characterized by two-dimensional sugar mapping analysis, while considering the structures of the N-linked chains of the zona protein mixture reported previously. Acidic complex-type chains were found at all three N-glycosylation sites, while Man5GlcNAc2 was found at Asn83 and Asn191, but there was very little of this sperm-ligand active chain at Asn527 in the ZP domain of ZPA.     

Antibody variable region glycosylation: position effects on antigen binding and carbohydrate structure.

The presence of N-linked carbohydrate at Asn58 in the VH of the antigen binding site of an antibody specific for alpha(1----6)dextran (TKC3.2.2) increases its affinity for dextran 10- to 50-fold. Site-directed mutagenesis has now been used to create novel carbohydrate addition sequences in the CDR2 of a non-glycosylated anti-dextran at Asn54 (TST2) and Asn60 (TSU7). These antibodies are glycosylated and the carbohydrates are accessible for lectin binding. The amino acid change in TSU7 (Lys62----Thr62) decreases the affinity for antigen; however, glycosylation of TSU7 increased its affinity for antigen 3-fold, less than the greater than 10-fold increase in affinity seen for glycosylated TKC3.2.2. The difference in impact of glycosylation could result either from the position of the carbohydrate or from its structure; unlike the other antibodies, TSU7 attaches a high mannose, rather than complex, carbohydrate in CDR2. In contrast, glycosylation of TST2 at amino acid 54 inhibits dextran binding. Thus slight changes in the position of the N-linked carbohydrate in the CDR2 of this antibody result in substantially different effects on antigen binding. Unlike what was observed for the anti-dextrans, a carbohydrate addition site placed in a similar position in an anti-dansyl is not utilized.     

The relevance of N-linked glycosylation to the binding of a ligand to guanylate cyclase C.

The role of carbohydrate moieties at the N-linked glycosylation sites of guanylate cyclase C (GC-C), a receptor protein for guanylin, uroguanylin and heat-stable enterotoxin, in ligand binding and structural stability was examined using site-directed mutagenesis of the putative N-linked glycosylation sites in the extracellular domain (ECD) of porcine GC-C. For this purpose, eight mutant proteins of ECD (N9A, N20A, N56A, N172A, N261A, N284A, N334A and N379A) and six mutant proteins of the complete GC-C (N9A, S11A, N172A, T174A, N379A and T381A) were prepared, in which Ala replaced Asn, Ser and Thr at the N-linked glycosylation consensus sites. All the mutant proteins showed a ligand-binding affinity (K(d)) similar to those of the wild-type proteins, although the deletion of a carbohydrate moiety at each of the N-linked glycosylation sites affected the ligand-binding ability of ECD or GC-C to some degree. However, the mutant proteins of ECD (N379A) and GC-C (N379A and T381A) showed considerably decreased binding ability in the context of maximum capacity (B(max)) to a ligand, despite the fact that the expression levels of these mutant proteins were nearly the same as the wild-type proteins. Moreover, the mutant protein of ECD (N379A) was considerably less stable to a denaturant. These results clearly indicate a crucial role for the carbohydrate moiety at N379, which is located near the transmembrane region, in structural stability, the ability to bind to a ligand and the cyclase catalytic activity of GC-C, and provide a route for the elucidation of the mechanism of the interaction between GC-C and a ligand.     

Regulation of receptor binding affinity of influenza virus hemagglutinin by its carbohydrate moiety.

The hemagglutinin (HA) of the fowl plague virus (FPV) strain of influenza A virus has two N-linked oligosaccharides attached to Asn123 and Asn149 in the vicinity of the receptor binding site. The effect of these carbohydrate side chains on the binding of HA to neuraminic acid-containing receptors has been analyzed. When the oligosaccharides were deleted by site-specific mutagenesis, HA expressed from a simian virus 40 vector showed enhanced hemadsorbing activity. Binding was so strong under these conditions that erythrocytes were no longer released by viral neuraminidase and that release was significantly reduced when neuraminidase from Vibrio cholerae was used. Similarly, when these oligosaccharides were removed selectively from purified viruses by N-glycosidase F, such virions were unable to elute from receptors, although they retained neuraminidase activity. Thus, release of FPV from cell receptors depends on the presence of the HA glycans at Asn123 and Asn149. On the other hand, receptor binding was abolished when these oligosaccharides were sialylated after expression in the absence of neuraminidase (M. Ohuchi, A. Feldmann, R. Ohuchi, and H.-D. Klenk, Virology 212:77-83, 1995). These observations indicate that the receptor affinity of FPV HA is controlled by oligosaccharides adjacent to the receptor binding site.     

Glycosylation is important for binding to human calcitonin receptors

Human calcitonin receptor (hCTR) subtypes contain three or four potential Asn-linked glycosylation sites in their extracellular amino termini. The role of glycosylation in hCTR function has not been identified, but it has been suggested that inhibition of glycosylation does not affect binding or signaling. To determine the role of glycosylation in hCTR biology, we studied the effects of inhibition of glycosylation and of substitution of Asn residues that are potential glycosylation sites. Native and mutated hCTRs were studied after transient expression in monkey kidney COS-1 cells. Tunicamycin, administered as part of a treatment protocol that inhibited glycosylation of all expressed receptors, decreased salmon calcitonin (sCT) binding affinities and signaling potencies at hCTRs with three or four potential glycosylation sites. In hCTR3, which contains three potential glycosylation sites at positions 26, 78, and 83, site-specific substitution of Asn-26 by Ala had no effect on sCT binding affinity or potency, whereas substitution of Asn-78 or Asn-83 lowered sCT affinity and potency. A mutant hCTR3 in which all three Asn residues were substituted with Ala exhibited no high-affinity sCT binding and potencies of several calcitonin analogues that were more than 100-fold lower than that of native hCTR3. Our data show that glycosylation is important for high-affinity binding and potency of calcitonin analogues at hCTRs.     

N-linked glycosylation of a beetle (Apriona germari) cellulase Ag-EGase II is necessary for enzymatic activity

We previously reported that the beta-1,4-endoglucanase (EGase) belonging to glycoside hydrolase family (GHF) 45 of the mulberry longicorn beetle, Apriona germari (Ag-EGase II), has three potential N-linked glycosylation sites; these sites are located at amino acid residues 56-59 (NKSG), 99-102 (NSTF), and 237-239 (NYSstop). In the present study, we analyze the functional role of these potential N-linked glycosylation sites. Tunicamycin treatment completely abolished the enzymatic activity of Ag-EGase II. To further elucidate the functional role of the N-linked glycosylation sites in Ag-EGase II, we have assayed the cellulase enzyme activity in Ser58Gln, Thr101Gln, or Ser239Gln mutants. Lack of N-linked glycosylation site at residues 99-102 (NSTF), the site of which is conserved in known beetle GHF 45 cellulases, showed loss of enzyme activity and reduced the molecular mass of the enzyme. In contrast, mutations in Ser58Gln or Ser239Gln affected neither the activity nor the apparent molecular mass of the enzyme, indicating that these sites did not lead to N-linked glycosylation. The present study demonstrates that N-linked glycosylation at residues 99-102 (NSTF), while not essential for secretion, is required for Ag-EGase II enzyme activity.     

Asparagine-linked glycosylation of human chymotrypsin C is required for folding and secretion but not for enzyme activity

Human chymotrypsin C (CTRC) plays a protective role in the pancreas by mitigating premature trypsinogen activation through degradation. Mutations that abolish activity or secretion of CTRC increase the risk for chronic pancreatitis. The aim of the present study was to determine whether human CTRC undergoes asparagine-linked (N-linked) glycosylation and to examine the role of this modification in CTRC folding and function. We abolished potential sites of N-linked glycosylation (Asn-Xaa-Ser/Thr) in human CTRC by mutating the Asn residues to Ser individually or in combination, expressed the CTRC mutants in HEK 293T cells and determined their glycosylation state using PNGase F and endo H digestion. We found that human CTRC contains a single N-linked glycan on Asn52. Elimination of N-glycosylation by mutation of Asn52 (N52S) reduced CTRC secretion about 10-fold from HEK 293T cells but had no effect on CTRC activity or inhibitor binding. Overexpression of the N52S CTRC mutant elicited endoplasmic reticulum stress in AR42J acinar cells, indicating that N-glycosylation is required for folding of human CTRC. Despite its important role, Asn52 is poorly conserved in other mammalian CTRC orthologs, including the rat which is monoglycosylated on Asn90. Introduction of the Asn90 site in a non-glycosylated human CTRC mutant restored full glycosylation but only partially rescued the secretion defect. We conclude that N-linked glycosylation of human CTRC is required for efficient folding and secretion; however, the N-linked glycan is unimportant for enzyme activity or inhibitor binding. The position of the N-linked glycan is critical for optimal folding, and it may vary among the otherwise highly homologous mammalian CTRC sequences.