Characterizing the Link between Glycosylation State and Enzymatic Activity of the Endo-β1,4-glucanase KORRIGAN1 from Arabidopsis thaliana

Defects in N-glycosylation and N-glycan processing frequently cause alterations in plant cell wall architecture, including changes in the structure of cellulose, which is the most abundant plant polysaccharide. KORRIGAN1 (KOR1) is a glycoprotein enzyme with an essential function during cellulose biosynthesis in Arabidopsis thaliana. KOR1 is a membrane-anchored endo-β1,4-glucanase and contains eight potential N-glycosylation sites in its extracellular domain. Here, we expressed A. thaliana KOR1 as a soluble, enzymatically active protein in insect cells and analyzed its N-glycosylation state. Structural analysis revealed that all eight potential N-glycosylation sites are utilized. Individual elimination of evolutionarily conserved N-glycosylation sites did not abolish proper KOR1 folding, but mutations of Asn-216, Asn-324, Asn-345, and Asn-567 resulted in considerably lower enzymatic activity. In contrast, production of wild-type KOR1 in the presence of the class I α-mannosidase inhibitor kifunensine, which abolished the conversion of KOR1 N-glycans into complex structures, did not affect the activity of the enzyme. To address N-glycosylation site occupancy and N-glycan composition of KOR1 under more natural conditions, we expressed a chimeric KOR1-Fc-GFP fusion protein in leaves of Nicotiana benthamiana. Although Asn-108 and Asn-133 carried oligomannosidic N-linked oligosaccharides, the six other glycosylation sites were modified with complex N-glycans. Interestingly, the partially functional KOR1 G429R mutant encoded by the A. thaliana rsw2-1 allele displayed only oligomannosidic structures when expressed in N. benthamiana, indicating its retention in the endoplasmic reticulum. In summary, our data indicate that utilization of several N-glycosylation sites is important for KOR1 activity, whereas the structure of the attached N-glycans is not critical.     

Role of complex N-glycans in plant stress tolerance

In plant cells, glycans attached to asparagine (N) residues of proteins undergo various modifications in the endoplasmic reticulum and the Golgi apparatus. The N-glycan modifications in the Golgi apparatus result in complex N-glycans attached to membrane proteins, secreted proteins and vacuolar proteins. Recently, we have investigated the role of complex N-glycans in plants using a series of Arabidopsis thaliana mutants affected in complex N-glycan biosynthesis.¹ Several mutant plants including complex glycan 1 (cgl1) displayed a salt-sensitive phenotype during their root growth, which was associated with radial swelling and loss of apical dominance. Among the proteins whose N-glycans are affected by the cgl1 mutation is a membrane anchored β1,4-endoglucanase, KORRIGAN1/RADIALLY SWOLLEN 2 (KOR1/RSW2) involved in cellulose biosynthesis. The cgl1 mutation strongly enhanced the phenotype of a temperature sensitive allele of KOR1/RSW2 (rsw2-1) even at the permissive temperature. This establishes that plant complex N-glycan modification is important for the in vivo function of KOR1/RSW2. Furthermore, rsw2-1 as well as another cellulose biosynthesis mutant rsw1-1 exhibited also a salt-sensitive phenotype at the permissive temperature. Based on these findings, we propose that one of the mechanisms that cause salt-induced root growth arrest is dysfunction of cell wall biosynthesis that induces mitotic arrest in the root apical meristem.Key words: Arabidopsis, salt stress, complex N-glycans, β1,4-endoglucanase, cell wallIn eukaryotic cells, both soluble and membrane proteins that enter the endoplasmic reticulum (ER) system may undergo post-translational modifications called N-glycosylation. N-glycosylation occurs in two phases, namely, core glycosylation in the ER and glycan maturation in the Golgi apparatus.^2,3 The process and roles of core glycosylation in the ER are well established and ubiquitous for eukaryotes. In the ER, pre-assembled core oligosaccharides (Glc₃Man₉GlcNac₂) are transferred to asparagine residues of the Asn-X-Ser/Thr motives in nascent polypeptides by the function of an oligosaccharyltransferase complex (OST). Terminal glucose residues are recognition sites for ER chaperones calnexin and calreticulin, and thus core N-glycans in the ER function in correct folding of newly synthesized proteins.^2,3Greater diversity exists in the N-glycan maturation steps in the Golgi apparatus and conspicuous roles for the resulting complex N-glycans.^2,4 In general, mature N-glycan structures are classified as oligomannosidic type, hybrid or complex type. Glycoprotein precursors that are exported from the ER carry high-mannose type N-glycan intermediates. Numerous enzymes are involved in the conversion of high-mannose type N-glycans to mature complex N-glycans. The functions of N-glycan modifications in the Golgi apparatus are well established in humans, because lack of N-glycan maturation results in Type II Congenital Disorders of Glycosylation.⁵ In Drosophila melanogaster, the Golgi pathway is necessary for development and function of the central nervous system,⁶ whereas in Candida albicans, it is necessary for cell wall integrity and virulence.⁷The first Arabidopsis thaliana mutant lacking complex N-glycans was reported in 1993.⁸ Since then, several mutants and transgenic plants altered in N-glycan maturation in the Golgi apparatus have been reported.^9–12 Plants with altered N-glycan modification pathways that are devoid of potentially immunogenic complex N-glycans are used for the production of pharmaceutical proteins^12,13 and could serve as potential food crops with reduced allergenicity. Until recently, however, plant complex N-glycans have not been associated with essential biological functions in their host plants due to lack of obvious phenotypes of mutant plants defective in complex N-glycan biosynthesis. We recently reported that mutants defective in complex N-glycans show enhanced salt sensitivity, establishing that complex N-glycans are indispensable for certain biological functions.¹Our previous study using an OST subunit mutant stt3a indicated that protein glycosylation could affect salt tolerance and root growth of A. thaliana.¹⁴ Since OST functions upstream of protein folding processes in the ER, stt3a caused an unfolded protein response (UPR), which is a general ER stress response to protein folding defects, as well as accumulation of under-glycosylated proteins. In our recent study, we tried to address whether the salt stress response of the mutant is caused by an activation of UPR, or by a shortage of functional glycoproteins produced by the cells.¹ The cgl1 mutant is defective in N-acetylglucosaminyltransferase in the Golgi apparatus¹⁵ and only able to produce oligomannosidic-type N-glycans but not complex-type N-glycans.⁸ cgl1 mutants exposed to salt stress exhibited root growth arrest and radial swelling similar to stt3a mutants, however, unlike stt3a, the cgl1 mutation did not cause UPR as judged by expression of an UPR marker gene, BiP_pro-GUS. This indicated that salt sensitivity of cgl1 (and likely also of stt3a) is due to lack of mature N-glycans essential for functionality of certain glycoprotein(s).We have determined that a membrane-anchored β1,4-endoglucanase, KORRIGAN1/RADIAL SWELLING2 (KOR1/RSW2), which functions in cellulose biosynthesis, is a target of CGL1 and involved in the salt stress response of A. thaliana.¹ A temperature sensitive rsw2-1 allele¹⁶ showed specific genetic interaction with both cgl1 and stt3a mutations. The corresponding double mutants exhibited spontaneous growth defects at the permissive temperature that were reminiscent of those of rsw2-1 at the restrictive temperature, of cgl1 and stt3a plants treated with salt, and of the rsw1-1 rsw2-1 double mutant that combines two cellulose deficiency mutations. This showed that cgl1 and stt3a enhance cellulose deficiency of rsw2-1, and in turn indicate that the KOR1/RSW2 protein requires complex N-glycans for its function in vivo. Further pyramiding of these mutations resulted in incremental enhancement of growth defects as well as developmental defects of the host plants (Kang et al., (2008), and Fig. 1). Importance of functional cellulose biosynthesis for salt tolerance was further supported by the novel finding of increased salt-sensitivity of rsw2-1 and rsw1-1 single mutants.¹Our previous and current data have implications that affect our view of protein N-glycosylation in plants. First, after all, plant complex N-glycans confer important in vivo functions to secreted/secretory glycoproteins, i.e., protect root growth from salt/osmotic stress. In contrast to core oligosaccharides in the ER, which globally affect protein folding, complex N-glycans appear to function at the individual protein level. Second, one of the targets of salt/osmotic stress is a component of the cellulose biosynthesis machinery, namely KOR1/RSW2 that requires complex N-glycans for its function. KOR1/RSW2 provides a link to how complex N-glycans protect plants from salt/osmotic stress. However, the mechanism by which salt stress triggers the growth arrest via KOR1/RSW2 dysfunction is not yet understood. We have previously shown that the root apical meristem of stt3a exhibits cell cycle arrest under salt stress, but cell differentiation and lateral root formation continued in the same root tip.¹⁴ This implies that plants, in response to salt stress and compromised cell-wall biosynthesis at the root apical meristem, specifically attenuate cell cycle progression at the old meristem and initiate new meristems. A signal transduction pathway that coordinates cell-wall integrity and cell proliferation is well documented in Sacchromyces cerevisiae, where Protein kinase C1 (Pkc1) and a MAP kinase cascade play essential roles.17 Interestingly, both S. cerevisiae Stt3 and Och1 (a mannosyltransferase in the Golgi apparatus) are involved in the cell-wall integrity pathway.¹⁷ In A. thaliana, mutations in the receptor kinase THESEUS1 suppressed hypocotyl elongation defects and ectopic lignification in several cellulose deficient mutants.¹⁸ However, since THE1 is expressed in elongation zones but not in cell division zones of root tips, and the1 did not suppress the kor1-1 phenotype,¹⁸ it is unlikely that THE1 is involved in the regulation of the salt stress response at the root apical meristem. This implies that dividing cells and expanding cells employ distinct mechanism to sense cellulose deficiency. Understanding how complex N-glycans regulate cell-wall biosynthesis and cell proliferation is an exciting task for the coming years.? Open in a separate windowFigure 1Scanning electron micrograph of one-week-old wild type (A and D), rsw2-1 stt3a-2 cgl1-T (B and E) and rsw2-1 rsw1-1 stt3a-2 cgl1-T (C and F) seedlings grown at 18°C. Severe growth defects in mutants are obvious. In shoot apical meristem (D–F), aberrant trichome development is seen in rsw2-1 stt3a-2 cgl1-T (E). In rsw2-1 rsw1-1 stt3a-2 cgl1-T (F), the meristem is transformed into unorganized mass of cells. Bars indicate 0.5 mm.     

Modification of N-glycosylation modulates the secretion and lipolytic function of apoptosis inhibitor of macrophage (AIM)

The mouse macrophage-derived apoptosis inhibitor of macrophage (AIM), which is incorporated into adipocytes and induces lipolysis by suppressing fatty acid synthase (FAS) activity, possesses three potential N-glycosylation sites. Inactivation of N-glycosylation sites revealed that mouse AIM contains two N-glycans in the first and second scavenger receptor cysteine-rich domains, and that depletion of N-glycans decreased AIM secretion from producing cells. Interestingly, the lack of N-glycans increased AIM lipolytic activity through enhancing AIM incorporation into adipocytes. Although human AIM contains no N-glycan, attachment of N-glycans increased AIM secretion. Thus, the N-glycosylation plays important roles in the secretion and lipolytic function of AIM.

Structured summary of protein interactions

AIMphysically interacts with FAS by anti tag coimmunoprecipitation (View interaction)     

N-Glycosylation and N-Glycan Moieties of CTB Expressed in Rice Seeds

Cholera toxin B subunit (CTB) is widely used as a carrier molecule and mucosal adjuvant and for the expression of fusion proteins of interest. CTB-fusion proteins are also expressed in plants, but the N-glycan structures of CTB have not been clarified. To gain insights into the N-glycosylation and N-glycans of CTB expressed in plants, we expressed CTB in rice seeds with an N-terminal glutelin signal and a C-terminal KDEL sequence and analyzed its N-glycosylation and N-glycan structures. CTB was successfully expressed in rice seeds in two forms: a form with N-glycosylation at Asn32 that included both plant-specific N-glycans and small oligomannosidic N-glycans and a non-N-glycosylated form. N-Glycan analysis of CTB showed that approximately 50 % of the N-glycans had plant-specific M3FX structures and that almost none of the N-glycans was of high-mannose-type N-glycan even though the CTB expressed in rice seeds contains a C-terminal KDEL sequence. These results suggest that the CTB expressed in rice was N-glycosylated through the endoplasmic reticulum (ER) and Golgi N-glycosylation machinery without the ER retrieval.     

N-glycan of ErbB family plays a crucial role in dimer formation and tumor promotion

More and more evidence indicates that N-glycan regulates signal transduction by modulating receptor functions. Previous studies suggested that glycosylation of EGFR is involved in dimerization and endocytosis. We further determined the role of N-glycosylation of ErbB family. A series of human ErbB3 mutants that lack each of the 10 N-glycosylation sites were prepared and transfected to Flp-In-CHO cells for stable expression. A crosslinking study showed that Asn 418 to Gln mutant (N418Q) of ErbB3 underwent autodimerization without its ligand, heregulin, and the heterodimer formation with ErbB2 was also increased. The N418Q mutant of ErbB3 co-expressed with ErbB2 promoted downstream signaling, anchorage-independent cell growth and the tumor growth in athymic mice. These findings suggest that the specific N-glycan in domain III of ErbB family plays an essential role in regulating receptor dimerization and transforming activity. We assume that the N-glycans affect the conformation of ErbB family, which is crucial for their activity. Together with findings from other laboratories, it is suggested that N-glycosylation controls ErbB signaling by various mechanisms.     

Pattern Recognition Receptors Require N-Glycosylation to Mediate Plant Immunity

N-Glycans attached to the ectodomains of plasma membrane pattern recognition receptors constitute likely initial contact sites between plant cells and invading pathogens. To assess the role of N-glycans in receptor-mediated immune responses, we investigated the functionality of Arabidopsis receptor kinases EFR and FLS2, sensing bacterial translation elongation factor Tu (elf18) and flagellin (flg22), respectively, in N-glycosylation mutants. As revealed by binding and responses to elf18 or flg22, both receptors tolerated immature N-glycans induced by mutations in various Golgi modification steps. EFR was specifically impaired by loss-of-function mutations in STT3A, a subunit of the endoplasmic reticulum resident oligosaccharyltransferase complex. FLS2 tolerated mild underglycosylation occurring in stt3a but was sensitive to severe underglycosylation induced by tunicamycin treatment. EFR accumulation was significantly reduced when synthesized without N-glycans but to lesser extent when underglycosylated in stt3a or mutated in single amino acid positions. Interestingly, EFR^N143Q lacking a single conserved N-glycosylation site from the EFR ectodomain accumulated to reduced levels and lost the ability to bind its ligand and to mediate elf18-elicited oxidative burst. However, EFR-YFP protein localization and peptide:N-glycosidase F digestion assays support that both EFR produced in stt3a and EFR^N143Q in wild type cells correctly targeted to the plasma membrane via the Golgi apparatus. These results indicate that a single N-glycan plays a critical role for receptor abundance and ligand recognition during plant-pathogen interactions at the cell surface.     

In vivo deglycosylation of recombinant glycoproteins in tobacco BY-2 cells

Production of recombinant pharmaceutical glycoproteins has been carried out in multiple expression systems. However, N-glycosylation, which increases heterogeneity and raises safety concerns due to the presence of non-human residues, is usually not controlled. The presence and composition of N-glycans are also susceptible to affect protein stability, function and immunogenicity. To tackle these issues, we are developing glycoengineered Nicotiana tabacum Bright Yellow-2 (BY-2) cell lines through knock out and ectopic expression of genes involved in the N-glycosylation pathway. Here, we report on the generation of BY-2 cell lines producing deglycosylated proteins. To this end, endoglycosidase T was co-expressed with an immunoglobulin G or glycoprotein B of human cytomegalovirus in BY-2 cell lines producing only high mannose N-glycans. Endoglycosidase T cleaves high mannose N-glycans to generate single, asparagine-linked, N-acetylglucosamine residues. The N-glycosylation profile of the secreted antibody was determined by mass spectrometry analysis. More than 90% of the N-glycans at the conserved Asn297 site were deglycosylated. Likewise, extensive deglycosylation of glycoprotein B, which possesses 18 N-glycosylation sites, was observed. N-glycan composition of gB glycovariants was assessed by in vitro enzymatic mobility shift assay and proven to be consistent with the expected glycoforms. Comparison of IgG glycovariants by differential scanning fluorimetry revealed a significant impact of the N-glycosylation pattern on the thermal stability. Production of deglycosylated pharmaceutical proteins in BY-2 cells expands the set of glycoengineered BY-2 cell lines.     

N-Glycosylation at the SynCAM (Synaptic Cell Adhesion Molecule) Immunoglobulin Interface Modulates Synaptic Adhesion

Select adhesion molecules connect pre- and postsynaptic membranes and organize developing synapses. The regulation of these trans-synaptic interactions is an important neurobiological question. We have previously shown that the synaptic cell adhesion molecules (SynCAMs) 1 and 2 engage in homo- and heterophilic interactions and bridge the synaptic cleft to induce presynaptic terminals. Here, we demonstrate that site-specific N-glycosylation impacts the structure and function of adhesive SynCAM interactions. Through crystallographic analysis of SynCAM 2, we identified within the adhesive interface of its Ig1 domain an N-glycan on residue Asn⁶⁰. Structural modeling of the corresponding SynCAM 1 Ig1 domain indicates that its glycosylation sites Asn⁷⁰/Asn¹⁰⁴ flank the binding interface of this domain. Mass spectrometric and mutational studies confirm and characterize the modification of these three sites. These site-specific N-glycans affect SynCAM adhesion yet act in a differential manner. Although glycosylation of SynCAM 2 at Asn⁶⁰ reduces adhesion, N-glycans at Asn⁷⁰/Asn¹⁰⁴ of SynCAM 1 increase its interactions. The modification of SynCAM 1 with sialic acids contributes to the glycan-dependent strengthening of its binding. Functionally, N-glycosylation promotes the trans-synaptic interactions of SynCAM 1 and is required for synapse induction. These results demonstrate that N-glycosylation of SynCAM proteins differentially affects their binding interface and implicate post-translational modification as a mechanism to regulate trans-synaptic adhesion.     

Engineering of Sialylated Mucin-type O-Glycosylation in Plants

Proper N- and O-glycosylation of recombinant proteins is important for their biological function. Although the N-glycan processing pathway of different expression hosts has been successfully modified in the past, comparatively little attention has been paid to the generation of customized O-linked glycans. Plants are attractive hosts for engineering of O-glycosylation steps, as they contain no endogenous glycosyltransferases that perform mammalian-type Ser/Thr glycosylation and could interfere with the production of defined O-glycans. Here, we produced mucin-type O-GalNAc and core 1 O-linked glycan structures on recombinant human erythropoietin fused to an IgG heavy chain fragment (EPO-Fc) by transient expression in Nicotiana benthamiana plants. Furthermore, for the generation of sialylated core 1 structures constructs encoding human polypeptide:N-acetylgalactosaminyltransferase 2, Drosophila melanogaster core 1 β1,3-galactosyltransferase, human α2,3-sialyltransferase, and Mus musculus α2,6-sialyltransferase were transiently co-expressed in N. benthamiana together with EPO-Fc and the machinery for sialylation of N-glycans. The formation of significant amounts of mono- and disialylated O-linked glycans was confirmed by liquid chromatography-electrospray ionization-mass spectrometry. Analysis of the three EPO glycopeptides carrying N-glycans revealed the presence of biantennary structures with terminal sialic acid residues. Our data demonstrate that N. benthamiana plants are amenable to engineering of the O-glycosylation pathway and can produce well defined human-type O- and N-linked glycans on recombinant therapeutics.     

Role of E-cadherin N-glycosylation profile in a mammary tumor model

Modifications in cell surface glycosylation affecting cell adhesion are common characteristics of transformed cells. This study characterizes the N-glycosylation profile of E-cadherin in models of canine mammary gland adenoma and carcinoma evaluating the importance of these glycosylation modifications in the malignant phenotype.Our results show that the pattern of E-cadherin N-glycosylation in mammary carcinoma is characterized by highly branched N-glycans, increase in sialylation and an expression of few high mannose structures. Detailed mass spectrometry analysis demonstrated a new N-glycosylation site containing a potential complex type N-glycan in E-cadherin from a mammary carcinoma cell line.Our study demonstrates the importance of E-cadherin N-glycans in the process of tumor development and in the transformation to the malignant phenotype.     

Several N-Glycans on the HIV Envelope Glycoprotein gp120 Preferentially Locate Near Disulphide Bridges and Are Required for Efficient Infectivity and Virus Transmission

The HIV envelope glycoprotein gp120 contains nine disulphide bridges and is highly glycosylated, carrying on average 24 N-linked glycans. Using a probability calculation, we here demonstrate that there is a co-localization of disulphide bridges and N-linked glycans in HIV-1 gp120, with a predominance of N-linked glycans in close proximity to disulphide bridges, at the C-terminal side of the involved cysteines. Also, N-glycans are frequently found immediately adjacent to disulphide bridges in gp120 at the N-terminal side of the involved cysteines. In contrast, N-glycans at positions close to, but not immediately neighboring disulphide bridges seem to be disfavored at the N-terminal side of the involved cysteines. Such a pronounced co-localization of disulphide bridges and N-glycans was also found for the N-glycans on glycoprotein E1 of the hepatitis C virus (HCV) but not for other heavily glycosylated proteins such as E2 from HCV and the surface GP from Ebola virus. The potential functional role of the presence of N-glycans near disulphide bridges in HIV-1 gp120 was studied using site-directed mutagenesis, either by deleting conserved N-glycans or by inserting new N-glycosylation sites near disulphide bridges. The generated HIV-1_NL4.3 mutants were subjected to an array of assays, determining the envelope glycoprotein levels in mutant viral particles, their infectivity and the capture and transmission efficiencies of mutant virus particles by DC-SIGN. Three N-glycans located nearby disulphide bridges were found to be crucial for the preservation of several of these functions of gp120. In addition, introduction of new N-glycans upstream of several disulphide bridges, at locations where there was a significant absence of N-glycans in a broad variety of virus strains, was found to result in a complete loss of viral infectivity. It was shown that the N-glycan environment around well-defined disulphide bridges of gp120 is highly critical to allow efficient viral infection and transmission.     

Distinct Roles of N-Glycosylation at Different Sites of Corin in Cell Membrane Targeting and Ectodomain Shedding

Corin is a membrane-bound protease essential for activating natriuretic peptides and regulating blood pressure. Human corin has 19 predicted N-glycosylation sites in its extracellular domains. It has been shown that N-glycans are required for corin cell surface expression and zymogen activation. It remains unknown, however, how N-glycans at different sites may regulate corin biosynthesis and processing. In this study, we examined corin mutants, in which each of the 19 predicted N-glycosylation sites was mutated individually. By Western analysis of corin proteins in cell lysate and conditioned medium from transfected HEK293 cells and HL-1 cardiomyocytes, we found that N-glycosylation at Asn-80 inhibited corin shedding in the juxtamembrane domain. Similarly, N-glycosylation at Asn-231 protected corin from autocleavage in the frizzled-1 domain. Moreover, N-glycosylation at Asn-697 in the scavenger receptor domain and at Asn-1022 in the protease domain is important for corin cell surface targeting and zymogen activation. We also found that the location of the N-glycosylation site in the protease domain was not critical. N-Glycosylation at Asn-1022 may be switched to different sites to promote corin zymogen activation. Together, our results show that N-glycans at different sites may play distinct roles in regulating the cell membrane targeting, zymogen activation, and ectodomain shedding of corin.     

Improvement of N-glycan site occupancy of therapeutic glycoproteins produced in Pichia pastoris

Yeast is capable of performing posttranslational modifications, such as N- or O-glycosylation. It has been demonstrated that N-glycans play critical biological roles in therapeutic glycoproteins by modulating pharmacokinetics and pharmacodynamics. However, N-glycan sites on recombinant glycoproteins produced in yeast can be underglycosylated, and hence, not completely occupied. Genomic homology analysis indicates that the Pichia pastoris oligosaccharyltransferase (OST) complex consists of multiple subunits, including OST1, OST2, OST3, OST4, OST5, OST6, STT3, SWP1, and WBP1. Monoclonal antibodies produced in P. pastoris show that N-glycan site occupancy ranges from 75–85 % and is affected mainly by the OST function, and in part, by process conditions. In this study, we demonstrate that N-glycan site occupancy of antibodies can be improved to greater than 99 %, comparable to that of antibodies produced in mammalian cells (CHO), by overexpressing Leishmania major STT3D (LmSTT3D) under the control of an inducible alcohol oxidase 1 (AOX1) promoter. N-glycan site occupancy of non-antibody glycoproteins such as recombinant human granulocyte macrophage colony-stimulating factor (rhGM-CSF) was also significantly improved, suggesting that LmSTT3D has broad substrate specificity. These results suggest that the glycosylation status of recombinant proteins can be improved by heterologous STT3 expression, which will allow for the customization of therapeutic protein profiles.     

Sequences from the First Fibronectin Type III Repeat of the Neural Cell Adhesion Molecule Allow O-Glycan Polysialylation of an Adhesion Molecule Chimera

Polysialic acid is a developmentally regulated, anti-adhesive polymer that is added to N-glycans on the fifth immunoglobulin domain (Ig5) of the neural cell adhesion molecule (NCAM). We found that the first fibronectin type III repeat (FN1) of NCAM is required for the polysialylation of N-glycans on the adjacent Ig5 domain, and we proposed that the polysialyltransferases recognize specific sequences in FN1 to position themselves for Ig5 N-glycan polysialylation. Other studies identified a novel FN1 acidic surface patch and α-helix that play roles in NCAM polysialylation. Here, we characterize the contribution of two additional FN1 sequences, Pro⁵¹⁰-Tyr⁵¹¹-Ser⁵¹² (PYS) and Gln⁵¹⁶-Val⁵¹⁷-Gln⁵¹⁸ (QVQ). Replacing PYS or the acidic patch dramatically decreases the O-glycan polysialylation of a truncated NCAM protein, and replacing the α-helix or QVQ shifts polysialic acid to FN1 O-glycans in full-length NCAM. We also found that the FN1 domain of the olfactory cell adhesion molecule, a homologous but unpolysialylated protein, could partially replace NCAM FN1. Inserting Pro⁵¹⁰-Tyr⁵¹¹ eliminated N-glycan polysialylation and enhanced O-glycosylation of an NCAM- olfactory cell adhesion molecule chimera, and inserting other FN1 sequences unique to NCAM, predominantly the acidic patch, created a new polysialyltransferase recognition site. Taken together, our results highlight the role of the FN1 α-helix and QVQ sequences in N-glycan polysialylation and demonstrate that the acidic patch primarily functions in O-glycan polysialylation.     

Cellulase Linkers Are Optimized Based on Domain Type and Function: Insights from Sequence Analysis,Biophysical Measurements,and Molecular Simulation

Cellulase enzymes deconstruct cellulose to glucose, and are often comprised of glycosylated linkers connecting glycoside hydrolases (GHs) to carbohydrate-binding modules (CBMs). Although linker modifications can alter cellulase activity, the functional role of linkers beyond domain connectivity remains unknown. Here we investigate cellulase linkers connecting GH Family 6 or 7 catalytic domains to Family 1 or 2 CBMs, from both bacterial and eukaryotic cellulases to identify conserved characteristics potentially related to function. Sequence analysis suggests that the linker lengths between structured domains are optimized based on the GH domain and CBM type, such that linker length may be important for activity. Longer linkers are observed in eukaryotic GH Family 6 cellulases compared to GH Family 7 cellulases. Bacterial GH Family 6 cellulases are found with structured domains in either N to C terminal order, and similar linker lengths suggest there is no effect of domain order on length. O-glycosylation is uniformly distributed across linkers, suggesting that glycans are required along entire linker lengths for proteolysis protection and, as suggested by simulation, for extension. Sequence comparisons show that proline content for bacterial linkers is more than double that observed in eukaryotic linkers, but with fewer putative O-glycan sites, suggesting alternative methods for extension. Conversely, near linker termini where linkers connect to structured domains, O-glycosylation sites are observed less frequently, whereas glycines are more prevalent, suggesting the need for flexibility to achieve proper domain orientations. Putative N-glycosylation sites are quite rare in cellulase linkers, while an N-P motif, which strongly disfavors the attachment of N-glycans, is commonly observed. These results suggest that linkers exhibit features that are likely tailored for optimal function, despite possessing low sequence identity. This study suggests that cellulase linkers may exhibit function in enzyme action, and highlights the need for additional studies to elucidate cellulase linker functions.     

The human CD10 lacking an N-glycan at Asn628 is deficient in surface expression and neutral endopeptidase activity

Background

CD10, also known as neprilysin or enkephalinase exhibiting neutral endopeptidase (NEP) activity, is expressed by B-lineage hematopoietic cells as well as a variety of cells from normal tissues. It cleaves peptides such as cytokines to act for terminating inflammatory responses. Although CD10 molecules of the human pre-B-cell line NALM-6 have 6 consensus N-glycosylation sites, three of them are known to be N-glycosylated by X-ray crystallography.

Methods

In order to investigate the role of N-glycans in the full expression of NEP activity, we modified N-glycans by treatment of NALM6 cells with various glycosidases or alter each of the consensus N-glycosylation sites by generating site-directed mutagenesis and compared the NEP activities of the sugar-altered CD10 with those of intact CD10.

Results

CD10 of the human B-cell line NALM-6 was dominantly localized in raft microdomains and heterogeneously N-glycosylated. Although neither desialylation nor further degalactosylation caused defective NEP activity, removal of only a small part of N-glycans by treatment with glycopeptidase F under non-denaturing conditions decreased NEP activity completely. All of the three consensus sites of CD10 in HEK293 cells introduced with wild type-CD10 were confirmed to be N-glycosylated. Surface expression of N-glycan at Asn₆₂₈-deleted CD10 by HEK293 cells was greatly decreased as well as it lost entire NEP activities.

Conclusions

N-glycosylation at Asn₆₂₈ is essential not only for NEP activities, but also for surface expression.

General significance

Quality control system does not allow dysfunctional ecto-type proteases to express on plasma membrane.     

Glycosylation of human plasma lipoproteins reveals a high level of diversity,which directly impacts their functional properties

AimsHuman plasma lipoproteins are known to contain various glycan structures whose composition and functional importance are starting to be recognized. We assessed N-glycosylation of human plasma HDL and LDL and the role of their glycomes in cellular cholesterol metabolism.MethodsN-glycomic profiles of native and neuraminidase-treated HDL and LDL were obtained using HILIC-UHPLC-FLD. Relative abundance of the individual chromatographic peaks was quantitatively expressed as a percentage of total integrated area and N-glycan structures present in each peak were elucidated by MALDI-TOF MS. The capacity of HDL to mediate cellular efflux of cholesterol and the capacity of LDL to induce cellular accumulation of cholesteryl esters were evaluated in THP-1 cells.ResultsHILIC-UHPLC-FLD analysis of HDL and LDL N-glycans released by PNGase F resulted in 22 and 18 distinct chromatographic peaks, respectively. The majority of N-glycans present in HDL (~70%) and LDL (~60%) were sialylated with one or two sialic acid residues. The most abundant N-glycan structure in both HDL and LDL was a complex type biantennary N-glycan with one sialic acid (A2G2S1). Relative abundances of several N-glycan structures were dramatically altered by the neuraminidase treatment, which selectively removed sialic acid residues. Native HDL displayed significantly greater efficacy in removing cellular cholesterol from THP-1 cells as compared to desialylated HDL (p < 0.05). Cellular accumulation of cholesteryl esters in THP-1 cells was significantly higher after incubations with desialylated LDL particles as compared to native LDL (p < 0.05).ConclusionsN-glycome of human plasma lipoproteins reveals a high level of diversity, which directly impacts functional properties of the lipoproteins.     

Arabidopsis thaliana KORRIGAN1 protein: N-glycan modification,localization, and function in cellulose biosynthesis and osmotic stress responses

Plant cellulose biosynthesis is a complex process involving cellulose-synthase complexes (CSCs) and various auxiliary factors essential for proper orientation and crystallinity of cellulose microfibrils in the apoplast. Among them is KORRIGAN1 (KOR1), a type-II membrane protein with multiple N-glycans within its C-terminal cellulase domain. N-glycosylation of the cellulase domain was important for KOR1 targeting to and retention within the trans-Golgi network (TGN), and prevented accumulation of KOR1 at tonoplasts. The degree of successful TGN localization of KOR1 agreed well with in vivo-complementation efficacy of the rsw2–1 mutant, suggesting non-catalytic functions in the TGN. A dynamic interaction network involving microtubules, CSCs, KOR1, and currently unidentified glycoprotein component(s) likely determines stress-triggered re-organization of cellulose biosynthesis and resumption of cell-wall growth under stress.     

ABH blood group antigens in N-glycan of human glycophorin A

We previously showed that a small proportion of the O-linked oligosaccharide chains of human glycophorin A (GPA) contains blood group A, B or H antigens, relevant to the ABO phenotype of the donor. The structures of these minor O-glycans have been established (Podbielska et al. (2004) [20]). By the use of immunochemical methods we obtained results indicating that ABH blood group epitopes are also present in N-glycan of human GPA (Podbielska and Krotkiewski (2000) [22]). In the present paper we report a detailed analysis of GPA N-glycans using nanoflow electrospray ionization tandem mass spectrometry. N-glycans containing A-, B- and H-related sequences were identified in GPA preparations obtained from erythrocytes of blood group A, B and O donors, respectively. The ABH blood group epitopes are present on one antenna of the N-glycan, whereas a known sialylated sequence NeuAcα2-6Galβ1-4GlcNAc- occurs on the other antenna and other details are in agreement with the known major structure of the GPA N-glycan. In the bulk of the biantennary sialylated N-glycans released from GPA preparations, the blood group ABH epitopes-containing N-glycans, similarly O-glycans, constituted only a minor part. The amount relative to other N-glycans was estimated to 2-6% of blood group H epitope-containing glycans released from GPA-O preparations and 1-2% of blood group A and B epitope-containing glycans, released from GPA-A and GPA-B, respectively.     

N-Glycosylation Regulates Fibroblast Growth Factor Receptor/EGL-15 Activity in Caenorhabditis elegans in Vivo

The regulation of cell function by fibroblast growth factors (FGFs) classically occurs through a dual receptor system of a tyrosine kinase receptor (FGFR) and a heparan sulfate proteoglycan co-receptor. Mutations in some consensus N-glycosylation sites in human FGFR result in skeletal disorders and craniosynostosis syndromes, and biophysical studies in vitro suggest that N-glycosylation of FGFR alters ligand and heparan sulfate binding properties. The evolutionarily conserved FGFR signaling system of Caenorhabditis elegans has been used to assess the role of N-glycosylation in the regulation of FGFR signaling in vivo. The C. elegans FGF receptor, EGL-15, is N-glycosylated in vivo, and genetic substitution of specific consensus N-glycosylation sites leads to defects in the maintenance of fluid homeostasis and differentiation of sex muscles, both of which are phenotypes previously associated with hyperactive EGL-15 signaling. These phenotypes are suppressed by hypoactive mutations in EGL-15 downstream signaling components or activating mutations in the phosphatidylinositol 3-kinase pathway, respectively. The results show that N-glycans negatively regulate FGFR activity in vivo supporting the notion that mutation of N-glycosylation sites in human FGFR may lead to inappropriate activation of the receptor.