Structural requirements for additional N-linked carbohydrate on recombinant human erythropoietin

N-Linked glycosylation is a post-translational event whereby carbohydrates are added to secreted proteins at the consensus sequence Asn-Xaa-Ser/Thr, where Xaa is any amino acid except proline. Some consensus sequences in secreted proteins are not glycosylated, indicating that consensus sequences are necessary but not sufficient for glycosylation. In order to understand the structural rules for N-linked glycosylation, we introduced N-linked consensus sequences by site-directed mutagenesis into the polypeptide chain of the recombinant human erythropoietin molecule. Some regions of the polypeptide chain supported N-linked glycosylation more effectively than others. N-Linked glycosylation was inhibited by an adjacent proline suggesting that sequence context of a consensus sequence could affect glycosylation. One N-linked consensus sequence (Asn123-Thr125) introduced into a position close to the existing O-glycosylation site (Ser126) had an additional O-linked carbohydrate chain and not an additional N-linked carbohydrate chain suggesting that structural requirements in this region favored O-glycosylation over N-glycosylation. The presence of a consensus sequence on the protein surface of the folded molecule did not appear to be a prerequisite for oligosaccharide addition. However, it was noted that recombinant human erythropoietin analogs that were hyperglycosylated at sites that were normally buried had altered protein structures. This suggests that carbohydrate addition precedes polypeptide folding.     

Conformation analysis of the N-glycosylation site Asn-X-Thr/Ser in glycoproteins

Theoretical conformational analysis of oligopeptides CH3CO-Asn-X-Thr-NHCH3 (X = Gly, Ala, Pro), modelling N-glycosylation site, and their glycosylated derivatives CH3CO-(GlcNAc beta 1-4GlcNAc beta 1) Asn-X-Thr-NHCH3 has been carried out. Active conformations of the site are found, corresponding to structural prerequisities of N-glycosylation: Asn residue's position in beta-turn and hydrogen bond formation between side chains of Asn and Thr/Ser residues. In this case the L conformation of the central residue X is most probable. Since Pro residue does not possess this conformation, sequences with X = Pro are not glycosylated. It is shown that glycosylation of the above-mentioned sites is accompanied by reorientation of the Asn residue's side chains.     

The effects of N-glycosylation sites and the N-terminal region on the biological function of beta1,3-N-acetylglucosaminyltransferase 2 and its secretion

Human beta1,3-N-acetylglucosaminyltransferase 2 (beta3GnT2) is thought to be an enzyme that extends the polylactosamine acceptor chains, but its function and structure analysis are unknown. To obtain insight into the structure of beta3GnT2, the effects of N-glycosylation on its biological function were evaluated using the addition of inhibitors, site-directed mutagenesis of potential N-glycosylation sites, and deletion of its N-terminal region using a fusion protein with GFP(uv) in a baculovirus expression system. Four of five potential N-glycosylation sites were found to be occupied, and their biological function and secretion were inhibited with the treatment of N-glycosylation inhibitor, tunicamycin. The N-glycosylation at Asn219 was necessary for the beta3GnT activity; moreover, N-glycosylation at Asn127 and Asn219 was critical for efficient protein secretion. When Ser221 was replaced with Thr, fusion protein was expressed as a single band, indicating that the double band of the expressed fusion protein was due to the heterogeneity of the glycosylation at Asn219. The truncated protein consisting of amino acids 82-397 (GFP(uv)-beta3GnT2Delta83), which lacked both one N-glycosylation site at Asn79 and the stem region of glycosyltransferase, was expressed as only a small form and showed no beta3GnT activity. These results suggest that the N-glycosylation site at Asn219, which is conserved throughout the beta1,3-glycosyltransferase family, is indispensable not only with regard to its biological function, but also to its secretion. The N-terminal region, which belongs to a stem region of glycosyltransferase, might also be important to the active protein structure.     

N-glycosylation at Asn(491) in the Asn-Xaa-Cys motif of human transferrin

Glycopeptides derived from human transferrin were exhaustively analyzed by matrix-assisted laser desorption ionization and electrospray ionization mass spectrometry (MS). Both MS techniques clearly revealed the sequences of and the attachment sites of bi-antennary complex-type oligosaccharides, at both Asn432 and Asn630, both of which are located in a well-known motif for N-glycosylation, Asn-Xaa-Ser/Thr, but also at Asn491 in the Asn-Xaa-Cys motif. The latter has been reported to be a minor N-glycosylation site in several glycoproteins. The relative abundance of this abnormal glycosylation was estimated to be approximately 2 mol% of the transferrin preparation used in this study.     

Site-specific glycosylation of human recombinant erythropoietin: analysis of glycopeptides or peptides at each glycosylation site by fast atom bombardment mass spectrometry

We have previously determined the carbohydrate structure of human recombinant erythropoietin [Sasaki, H., Bothner, B., Dell, A., & Fukuda, M. (1987) J. Biol. Chem. 262, 12059-12076]. The carbohydrate chains are distributed in three N-glycosylation sites and one O-glycosylation site. In order to examine the extent to which protein structure influences glycosylation, we have analyzed the saccharide structures at each glycosylation site (Asn24, Asn38, Asn83, and Ser126) of human recombinant erythropoietin. By high-performance liquid chromatography, we have succeeded in separation of glycopeptides containing different O-linked saccharides to the same peptide backbone. Fast atom bombardment mass spectrometry of the isolated glycopeptides combined with Edman degradation allowed us to elucidate the composition of glycopeptides and the amino acid attachment site. The analysis of glycopeptides and saccharides by fast atom bombardment mass spectrometry and high-performance liquid chromatography provided the following conclusions on N-glycans: (1) saccharides at Asn24 are heterogeneous and consist of biantennary, triantennary, and tetraantennary saccharides with or without N-acetyllactosaminyl repeats; (2) saccharides at Asn38 mainly consist of well-processed saccharides such as tetraantennary saccharides with or without N-acetyllactosaminyl repeats; (3) saccharides at Asn83, on the other hand, are homogeneous in the backbone structure and are composed mainly of tetraantennary without N-acetyllactosaminyl repeats. It was also noted that saccharides at Asn24 are much less sialylated than those at Asn38, although these two glycosylation sites are close to each other. These results clearly indicate that the protein structure and, possibly, the carbohydrate chain at the neighboring site greatly influence glycosylation of a given glycosylation site.     

Glycosylation of the calcitonin receptor-like receptor at Asn(60) or Asn(112) is important for cell surface expression

The human calcitonin (CT) receptor-like receptor (hCRLR) of the B family of G protein-coupled receptors is N-glycosylated and associates with receptor-activity-modifying proteins for functional interaction with CT gene-related peptide (CGRP) or adrenomedullin (ADM), respectively. Three putative N-glycosylation sites Asn(60), Asn(112) and Asn(117) are present in the amino-terminal extracellular domain of the hCRLR. Tunicamycin dose-dependently inhibited the glycosylation of a myc-tagged hCRLR and in parallel specific [(125)I]CGRP and -ADM binding. Similarly, the double mutant myc-hCRLR(N60,112T) exhibited minimal N-glycosidase F sensitive glycosylation, presumably at the third Asn(117), and the cell surface expression and specific radioligand binding were impaired. Substitution of the Asn(117) by Thr abolished CGRP and ADM binding in the face of intact N-glycosylation and cell surface expression.     

Exchange of Ser-4 for Val, Leu or Asn in the sequon Asn-Ala-Ser does not prevent N-glycosylation of the cell surface glycoprotein from Halobacterium halobium

The archaeon Halobacterium halobium expresses a cell surface glycoprotein (CSG) with a repeating pentasaccharide unit N- glycosidically linked via N-acetylgalactosamine to Asn-2 of the polypeptide (GalNAc(1-N)Asn linkage type). This aspar-agine of the linkage unit is located within the N-terminal sequence Ala-Asn-Ala-Ser- , in accordance with the tripeptide consensus sequence Asn-Xaa-Ser/Thr typical for nearly every N-glycosylation site known so far, which are of the GlcNAc(1-N)-Asn linkage type. By a gene replacement method csg mutants were created which replace the serine residue of the consensus sequence by valine, leucine, and asparagine. Unexpectedly, this elimination of the consensus sequence did not prevent N-glycosylation. All respective mutant cell surface glycoproteins were N-glycosylated at Asn-2 with the same N-glycan chain as the wild type CSG. Asn-479 is N- glyco-sylated via a Glc(1-N)Asn linkage type in the wild type CSG. Replacement of Ser-481 in the sequence Asn-Ser-Ser for valine prevented glycosylation of Asn-479. From these results we postulate the existence of two different N-glycosyltransferases in H.halobium, one of which does not use the typical consensus sequence Asn-Xaa-Ser/Thr necessary for all other N-glycosyltransferases described so far.      

N-glycosylation at noncanonical Asn-X-Cys sequences in plant cells

The vesicular transport pathway in plant cells is often used for higher accumulation of recombinant proteins. In the endoplasmic reticulum, which acts as a gateway to the vesicular transport pathway, N-glycosylation occurs on specific Asn residues. This N-glycosylation in recombinant proteins must be carefully regulated as it can impact their enzymatic activity, half lives in serum when injected, structural stability, etc. In eukaryotic cells, including plant cells, N-glycans were found to be attached to Asn residues in Asn-X-Ser/Thr (X ≠ Pro) sequences. However, recently, N-glycosylations at noncanonical Asn-X-Cys sequences have been found in mammals and yeast. Our laboratory has discovered that N-glycans are attached to Asn residues at Asn-Thr-Cys sequences of double-repeated B subunit of Shiga toxin 2e produced in plant cells, the first reported case of N-glycosylation at a noncanonical Asn-X-Cys sequence in plant cells.     

Asn64-glycosylation affects Hypocrea jecorina (syn. Trichoderma reesei) cellobiohydrolase Cel7A activity expressed in Pichia pastoris

In this study, four N-glycosylation sites, Asn45, Asn64, Asn270 and Asn384 of Hypocrea jecorina (syn. Trichoderma reesei) Cel7A (family 7 cellobiohydrolase I) were replaced by serines using site-directed mutagenesis. These four mutants and wild type H. jecorina Cel7A gene were transformed into P. pastoris, and the recombinant enzymes were purified and analyzed. The enzymatic activities of recombinant Cel7A (rCel7A), and mutants N45S, N270S and N384S were very low while mutant N64S displayed about seven times higher activity than that of rCel7A, and about 10% of the wild-type Cel7A activity from H. jecorina. The results indicate that N-glycosylation of Asn64 had an effect on the activity of the Cel7A enzyme expressed in P. pastoris, and that glycosylation at this site would be only a subordinate reason for the low activity of the recombinant enzyme.     

Role of the putative N-glycosylation and PKC-phosphorylation sites of the human sodium-dependent multivitamin transporter (hSMVT) in function and regulation

The sodium-dependent multivitamin transporter (SMVT) is a major biotin transporter in a variety of tissues including the small intestine. The human SMVT (hSMVT) polypeptide is predicted to have four N-glycosylation sites and two putative PKC phosphorylation sites but their role in the function and regulation of the protein is not known and was examined in this investigation. Our results showed that the hSMVT protein is glycosylated and that this glycosylation is important for its function. Studies utilizing site-directed mutagenesis revealed that the N-glycosylation sites at positions Asn(138) and Asn(489) are important for the function of hSMVT and that mutating these sites significantly reduces the V(max) of the biotin uptake process. Mutating the putative PKC phosphorylation site Thr(286) of hSMVT led to a significant decrease in the PMA-induced inhibition in biotin uptake. The latter effect was not mediated via changes in the level of expression of the hSMVT protein and mRNA or in its level of expression at the cell membrane. These findings demonstrate that the hSMVT protein is glycosylated, and that glycosylation is important for its function. Furthermore, the study shows a role for the putative PKC-phosphorylation site Thr(286) of hSMVT in the PKC-mediated regulation of biotin uptake.     

Glycosylation sites and site-specific glycosylation in human Tamm- Horsfall glycoprotein

The N-glycosylation sites of human Tamm-Horsfall glycoprotein from one healthy male donor have been characterized, based on an approach using endoproteinase Glu-C (V-8 protease, Staphylococcus aureus ) digestion and a combination of chromatographic techniques, automated Edman sequencing, and fast atom bombardment mass spectrometry. Seven out of the eight potential N-glycosylation sites, namely, Asn52, Asn56, Asn208, Asn251, Asn298, Asn372, and Asn489, turned out to be glycosylated, and the potential glycosylation site at Asn14, being close to the N-terminus, is not used. The carbohydrate microheterogeneity on three of the glycosylation sites was studied in more detail by high-pH anion-exchange chromatographic profiling and 500 MHz1H-NMR spectroscopy. Glycosylation site Asn489 contains mainly di- and tri-charged oligosaccharides which comprise, among others, the GalNAc4 S (beta1-4)GlcNAc terminal sequence. Only glycosylation site Asn251 bears oligomannose-type carbohydrate chains ranging from Man5GlcNAc2to Man8GlcNAc2, in addition to a small amount of complex- type structures. Profiling of the carbohydrate moieties of Asn208 indicates a large heterogeneity, similar to that established for native human Tamm-Horsfall glycoprotein, namely, multiply charged complex-type carbohydrate structures, terminated by sulfate groups, sialic acid residues, and/or the Sda-determinant.      

Analysis of site-specific glycosylation in recombinant human follistatin expressed in Chinese hamster ovary cells.

Follistatin (FS), a glycoprotein, plays an important role in cell growth and differentiation through the neutralization of the biological activities of activins. In this study, we analyzed the glycosylation of recombinant human FS (rhFS) produced in Chinese hamster ovary cells. The results of SDS-PAGE and MALDI-TOF MS revealed the presence of both non-glycosylated and glycosylated forms. FS contains two potential N-glycosylation sites, Asn95 and Asn259. Using mass spectrometric peptide/glycopeptide mapping and precursor-ion scanning, we found that both N-glycosylation sites were partially glycosylated. Monosaccharide composition analyses suggested the linkages of fucosylated bi- and triantennary complex-type oligosaccharides on rhFS. This finding was supported by mass spectrometric oligosaccharide profiling, in which the m/z values and elution times of some of the oligosaccharides from rhFS were in good agreement with those of standard oligosaccharides. Site-specific glycosylation was deduced on the basis of the mass spectra of the glycopeptides. It was suggested that biantennary oligosaccharides are major oligosaccharides located at both Asn95 and Asn259, whereas the triantennary structures are present mainly at Asn95.     

N-glycosylation sites of plant purple acid phosphatases important for protein expression and secretion in insect cells

Analysis of plant purple acid phosphatases (PAPs) showed high conservation and different distribution of N-glycosylation sites. Oligosaccharide structures of Lupinus luteus acid phosphatase (Lu_AP) produced in insect cells were determined. Mutant Lu_AP and Phaseolus vulgaris (Ph_AP) phosphatases lacking possibility of N-glycosylation at highly conserved sites were generated and expressed in insect cells. A role for N-glycosylation in the stability of PAPs was indicated by unsuccessful attempts to secrete Ph_AP and Lu_AP mutants generated by replacing Asn residues of conserved glycosylation sequons by Ser residues either singly or in combination. We showed that Ph_AP belongs to the group of glycoproteins that require occupancy of all highly conserved glycosylation sites for secretion, whereas replacing of the third position of the glycosylation sequon indicated that Lu_AP may tolerate the absence of some N-glycans. However, the N-glycan located at the polypeptide C-terminus was crucial for secretion of both enzymes. PAP specific activity of glycosylation mutants successfully secreted was similar to the wild-type recombinant proteins.     

N-Glycans mutations rule oligomeric assembly and functional expression of P2X3 receptor for extracellular ATP

N-Glycosylation affects the function of ion channels at the level of multisubunit assembly, protein trafficking, ligand binding and channel opening. Like the majority of membrane proteins, ionotropic P2X receptors for extracellular ATP are glycosylated in their extracellular moiety. Here, we used site-directed mutagenesis to the four predicted N-glycosylation sites of P2X(3) receptor (Asn(139), Asn(170), Asn(194) and Asn(290)) and performed comparative analysis of the role of N-glycans on protein stability, plasma membrane delivery, trimer formation and inward currents. We have found that in transiently transfected HEK293 cells, Asn(170) is apparently the most important site for receptor stability, since its mutation causes a primary loss in protein content and indirect failure in membrane expression, oligomeric association and inward current responses. Even stronger effects are obtained when mutating Thr(172) in the same glycosylation consensus. Asn(194) and Asn(290) are the most dispensable, since even their simultaneous mutation does not affect any tested receptor feature. All double mutants containing Asn(170) mutation or the Asn(139)/Asn(290) double mutant are instead almost unable to assemble into a functional trimeric structure. The main emerging finding is that the inability to assemble into trimers might account for the impaired function in P2X(3) mutants where residue Asn(170) is replaced. These results improve our knowledge about the role of N-glycosylation in proper folding and oligomeric association of P2X(3) receptor.     

N-glycosylation of the human melanocortin 1 receptor: occupancy of glycosylation sequons and functional role

The melanocortin 1 receptor (MC1R), a major determinant of skin pigmentation and phototype, mediates the actions of α-melanocyte-stimulating hormone on melanocytes and is critical for melanocyte proliferation and differentiation. MC1R has two putative N-glycosylation targets, Asn15 and Asn29. It has been shown that MC1R is a glycoprotein with an unusual sensitivity to endoglycosidase H digestion. However, the occupancy and functional importance of each specific glycosylation sequon remains unknown. We demonstrate that MC1R is N-glycosylated at Asn15 and Asn29, with structurally and functionally different glycan chains. N-glycosylation is not necessary for high affinity agonist binding or functional coupling but has a strong effect on the availability of MC1R molecules on the plasma membrane, most likely by a combination of improved forward trafficking and decreased internalization. Finally, we found that MC1R variants exhibit different degrees of glycosylation which do not show a simple correlation with their functional status or intracellular trafficking.     

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.     

Isolation of a glycosylated form of the chicken eggshell protein ovocleidin and determination of the glycosylation site. Alternative glycosylation/phosphorylation at an N-glycosylation sequon

Ovocleidin, a major protein of the avian eggshell calcified layer, occurs in the eggshell soluble organic matrix in at least two forms. The major form is a phosphoprotein with two phosphorylated serines (OC-17) which was sequenced recently. A minor form is a glycosylated protein with identical sequence and only one phosphorylated serine (OC-23). The site of glycosylation is Asn(59), the only asparagine in the amino acid sequence contained in the N-glycosylation site consensus sequence, N-A-S. Ser(61), which is part of this site, is phosphorylated in OC-17 but not in OC-23 indicating that the two modifications are mutually exclusive. This is the first example of alternative glycosylation/phosphorylation occurring at an N-glycosylation site.     

An analysis of amino acid sequences surrounding archaeal glycoprotein sequons

Despite having provided the first example of a prokaryal glycoprotein, little is known of the rules governing the N-glycosylation process in Archaea. As in Eukarya and Bacteria, archaeal N-glycosylation takes place at the Asn residues of Asn-X-Ser/Thr sequons. Since not all sequons are utilized, it is clear that other factors, including the context in which a sequon exists, affect glycosylation efficiency. As yet, the contribution to N-glycosylation made by sequon-bordering residues and other related factors in Archaea remains unaddressed. In the following, the surroundings of Asn residues confirmed by experiment as modified were analyzed in an attempt to define sequence rules and requirements for archaeal N-glycosylation.     

Database analysis of O-glycosylation sites in proteins

Statistical analysis was carried out to study the sequential aspects of amino acids around the O-glycosylated Ser/Thr. 992 sequences containing O-glycosylated Ser/Thr were selected from the O-GLYCBASE database of O-glycosylated proteins. The frequency of occurrence of amino acid residues around the glycosylated Ser/Thr revealed that there is an increased number of proline residues around the O-glycosylation sites in comparison with the nonglycosylated serine and threonine residues. The deviation parameter calculated as a measure of preferential and nonpreferential occurrence of amino acid residues around the glycosylation site shows that Pro has the maximum preference around the O-glycosylation site. Pro at +3 and/or -1 positions strongly favors glycosylation irrespective of single and multiple glycosylation sites. In addition, serine and threonine are preferred around the multiple glycosylation sites due to the effect of clusters of closely spaced glycosylated Ser/Thr. The preference of amino acids around the sites of mucin-type glycosylation is found likely to be similar to that of the O-glycosylation sites when taken together, but the acidic amino acids are more preferred around Ser/Thr in mucin-type glycosylation when compared totally. Aromatic amino acids hinder O-glycosylation in contrast to N-glycosylation. Cysteine and amino acids with bulky side chains inhibit O-glycosylation. The preference of certain potential sequence motifs of glycosylation has been discussed.     

The role of N-glycosylation sites in the activity,stability, and expression of the recombinant elastase expressed by Pichia pastoris

The Pseudomonas aeruginosa elastase (PAE), produced by Pseudomonas aeruginosa (P. aeruginosa), is a promising biocatalyst for peptide synthesis in organic solvents. As P. aeruginosa is an opportunistic pathogen, the enzyme has been heterologously over-expressed in the safe and efficient host, Pichia pastoris (P. pastoris) for its industrial application. The recombinant elastase (rPAE) contains three potential N-glycosylation sites (Asn-Xaa-Ser/Thr consensus sequences), and is heterogeneously N-glycosylated. To investigate the role of N-glycosylation in the activity, stability, and expression of rPAE, these potential N-glycosylation sites (N43, N212, and N280) were mutated using site-directed mutagenesis. Specifically the asparagine (Asn, N) residues were converted to glutamine (Gln, Q). The enzymatic activity and stability of non-glycosylated and glycosylated rPAE were then compared. The results indicated that the influence of N-glycosylation on its activity was insignificant. The non- and glycosylated isoforms of rPAE displayed similar kinetic parameters for hydrolyzing casein in aqueous medium, and when catalyzing bipeptide synthesis in 50% (v/v) DMSO, they exhibited identical substrate specificity and activity, and produced similar yields. However, N-glycosylation improved rPAE stability both in aqueous medium and in 50% (v/v) organic solvents. The half-lives of the glycosylated and non-glycosylated forms of rPAE at 70 °C were 32.2 and 23.1 min, respectively. Mutation of any potential N-glycosylation site was detrimental to its expression in P. pastoris. There was a 23.9% decrease in expression of the N43Q mutant, 63.6% of the N212Q mutant, and 63.7% of the N280Q mutant compared with the wild type. Furthermore, combined mutation of these sites resulted in an additional decrease in the caseinolytic activities of the mutants. These results indicated that all of the N-glycosylation sites were necessary for high-level expression of rPAE.