N-glycan Chain Research Articles

Endometrial regeneration cell-derived exosomes loaded with siSLAMF6 inhibit cardiac allograft rejection through the suppression of desialylation modification

BackgroundsAcute transplant rejection is a major component of poor prognoses for organ transplantation. Owing to the multiple complex mechanisms involved, new treatments are still under exploration. Endometrial regenerative cells (ERCs) have been widely used in various refractory immune-related diseases, but the role of ERC-derived exosomes (ERC-Exos) in alleviating transplant rejection has not been extensively studied. Signaling lymphocyte activation molecule family 6 (SLAMF6) plays an important role in regulating immune responses. In this study, we explored the main mechanism by which ERC-Exos loaded with siSLAMF6 can alleviate allogeneic transplant rejection.MethodsC57BL/6 mouse recipients of BALB/c mouse kidney transplants were randomly divided into four groups and treated with exosomes. The graft pathology was evaluated by H&E staining. Splenic and transplanted heart immune cell populations were analyzed by flow cytometry. Recipient serum cytokine profiles were determined by enzyme-linked immunosorbent assay (ELISA). The proliferation and differentiation capacity of CD4+ T cell populations were evaluated in vitro. The α-2,6-sialylation levels in the CD4+ T cells were determined by SNA blotting.ResultsIn vivo, mice treated with ERC-siSLAMF6 Exo achieved significantly prolonged allograft survival. The serum cytokine profiles of the recipients were significantly altered in the ERC-siSLAMF6 Exo-treated recipients. In vitro, we found that ERC-siSLAMF6-Exo considerably downregulated α-2,6-sialyltransferase (ST6GAL1) expression in CD4+ T cells, and significantly reduced α-2,6-sialylation levels. Through desialylation, ERC-siSLAMF6 Exo therapy significantly decreased CD4+ T cell proliferation and inhibited CD4+ T cell differentiation into Th1 and Th17 cells while promoting regulatory T cell (Treg) differentiation.ConclusionsOur study indicated that ERC-Exos loaded with siSLAMF6 reduce the amount of sialic acid connected to α-2,6 at the end of the N-glycan chain on the CD4+ T cell surface, increase the number of therapeutic exosomes endocytosed into CD4+ T cells, and inhibit the activation of T cell receptor signaling pathways, which prolongs allograft survival. This study confirms the feasibility of using ERC-Exos as natural carriers combined with gene therapy, which could be used as a potential therapeutic strategy to alleviate allograft rejection.

RNA-seq analysis identifies age-dependent changes in expression of mRNAs - encoding N-glycosylation pathway enzymes in mouse gonadotropes

Follicle-stimulating hormone (FSH) is a glycoprotein that is assembled as a heterodimer of α/β subunits in gonadotropes. Each subunit contains two N-glycan chains. Our previous in vivo genetic studies identified that at least one N-glycan chain must be present on the FSHβ subunit for efficient FSH dimer assembly and secretion. Moreover, macroheterogeneity observed uniquely on human FSHβ results in ratiometric changes in age-specific FSH glycoforms, particularly during menopausal transition. Despite the recognition of many prominent roles of sugars on FSH including dimer assembly and secretion, serum half-life, receptor binding and signal transduction, the N-glycosylation machinery in gonadotropes has never been defined. Here, we used a mouse model in which gonadotropes are GFP-labeled in vivo and achieved rapid purification of GFP+ gonadotropes from pituitaries of female mice at reproductively young, middle, and old ages. We identified by RNA-seq analysis 52 mRNAs encoding N-glycosylation pathway enzymes expressed in 3- and 8-10-month-old mouse gonadotropes. We hierarchically mapped and localized the enzymes to distinct subcellular organelles within the N-glycosylation biosynthetic pathway. Of the 52 mRNAs, we found 27 mRNAs are differentially expressed between the 3- and 8-10-month old mice. We subsequently selected 8 mRNAs which showed varying changes in expression for confirmation of abundance in vivo via qPCR analysis, using more expanded aging time points with distinct 8-month and 14-month age groups. Real time qPCR analysis indicated dynamic changes in expression of N-glycosylation pathway enzyme-encoding mRNAs across the life span. Notably, computational analysis predicted the promoters of genes encoding these 8 mRNAs contain multiple high probability binding sites for estrogen receptor-1 and progesterone receptor. Collectively, our studies define the N-glycome and identify age-specific dynamic changes in mRNAs encoding N-glycosylation pathway enzymes in mouse gonadotropes. Our studies suggest the age-related decline in ovarian steroids may regulate expression of N-glycosylation enzymes in mouse gonadotropes and explain the age-related N-glycosylation shift previously observed on human FSHβ subunit in pituitaries of women.

Core Fucosylation Is Required for the Secretion of and the Enzymatic Activity of SOD3 in Nonsmall-Cell Lung Cancer Cells.

Aims: The anticancer function of superoxide dismutases (SODs) is still controversial. SOD3 is an extracellular superoxide dismutase and contains a single N-glycan chain. The role played by the N-glycosylation of SOD3, as it relates to lung cancer, is poorly understood. For this, we performed the structural and functional analyses of the N-glycan of SOD3 in lung cancer. Results: We report herein that the fucose structure of the N-glycan in SOD3 was increased in the sera of patients with lung cancer. In cell lines of non-small lung cancer cell (NSCLC), we also found a high level of the core fucose structure in the N-glycan of SOD3, as determined by lectin blotting and mass spectrometry analysis. To address the roles of the core fucose structure of SOD3, we generated FUT8 (α1,6-fucosyltransferase) gene knockout A549 cells. Using these cells, we found that the core fucose structure of SOD3 was required for its secretion and enzymatic activity, which contributes to the suppression of cell growth of NSCLC cells. Innovation: The core fucosylation is required for the secretion and enzymatic activity of SOD3, which contributes to anti-tumor functions such as the suppression of cell growth of NSCLC. Conclusion: The N-glycans, especially those with core fucose structures, regulate the anti-tumor functions of SOD3 against NSCLC. Antioxid. Redox Signal. 38, 1201-1211.

Dissecting the essential role of N-glycosylation in catalytic performance of xanthan lyase

Modified xanthan produced by xanthan lyase has broad application prospects in the food industry. However, the catalytic performance of xanthan lyase still needs to be improved through rational design. To address this problem, in this work, the glycosylation and its influences on the catalytic performance of a xanthan lyase (EcXly), which was heterologously expressed in Escherichia coli, were reported. Liquid chromatography coupled to tandem mass spectrometry analysis revealed that the N599 site of EcXly was modified by a single N-glycan chain. Based on sequence alignment and three-dimensional structure prediction, it could be deduced that the N599 site was located in the catalytic domain of EcXly and in close proximity to the catalytic residues. After site-directed mutagenesis of N599 with alanine, aspartic acid and glycine, respectively, the EcXly and its mutants were characterized and compared. The results demonstrated that elimination of the N-glycosylation had diminished the specific activity, pH stability, and substrate affinity of EcXly. Fluorescence spectra further revealed that the glycosylation could significantly affect the overall tertiary structure of EcXly. Therefore, in prokaryotic hosts, the N-glycosylation could influence the catalytic performance of the enzyme by changing its structure. To the best of our knowledge, this is the first report about the post-translational modification of xanthan lyase in prokaryotes. Overall, our work enriched research on the role of glycan chains in the functional performance of proteins expressed in prokaryotes and should be valuable for the rational design of xanthan lyase to produce modified xanthan for industrial application.Graphical

Different N-Glycosylation Sites Reduce the Activity of Recombinant DSPAα2.

Bat plasminogen activators α2 (DSPAα2) has extremely high medicinal value as a powerful natural thrombolytic protein. However, wild-type DSPAα2 has two N-glycosylation sites (N185 and N398) and its non-human classes of high-mannose-type N-glycans may cause immune responses in vivo. By mutating the N-glycosylation sites, we aimed to study the effect of its N-glycan chain on plasminogen activation, fibrin sensitivity, and to observe the physicochemical properties of DSPAα2. A logical structure design was performed in this study. Four single mutants and one double mutant were constructed and expressed in Pichia pastoris. When the N398 site was eliminated, the plasminogen activator in the mutants had their activities reduced to ~40%. When the N185 site was inactivated, there was a weak decrease in the plasminogen activation of its mutant, while the fibrin sensitivity significantly decreased by ~10-fold. Neither N-glycosylation nor deglycosylation mutations changed the pH resistance or heat resistance of DSPAα2. This study confirms that N-glycosylation affects the biochemical function of DSPAα2, which provides a reference for subsequent applications of DSPAα2.

Monoallelic pathogenic ALG5 variants cause atypical polycystic kidney disease and interstitial fibrosis

Disorders of the autosomal dominant polycystic kidney disease (ADPKD) spectrum are characterized by the development of kidney cysts and progressive kidney function decline. PKD1 and PKD2, encoding polycystin (PC)1 and 2, are the two major genes associated with ADPKD; other genes include IFT140, GANAB, DNAJB11, and ALG9. Genetic testing remains inconclusive in ∼7% of the families. We performed whole-exome sequencing in a large multiplex genetically unresolved (GUR) family affected by ADPKD-like symptoms and identified a monoallelic frameshift variant (c.703_704delCA) in ALG5. ALG5 encodes an endoplasmic-reticulum-resident enzyme required for addition of glucose molecules to the assembling N-glycan precursors. To identify additional families, we screened a cohort of 1,213 families with ADPKD-like and/or autosomal-dominant tubulointerstitial kidney diseases (ADTKD), GUR (n= 137) or naive to genetic testing (n= 1,076), by targeted massively parallel sequencing, and we accessed Genomics England 100,000 Genomes Project data. Four additional families with pathogenic variants in ALG5 were identified. Clinical presentation was consistent in the 23 affected members, with non-enlarged cystic kidneys and few or no liver cysts; 8 subjects reached end-stage kidney disease from 62 to 91 years of age. We demonstrate that ALG5 haploinsufficiency is sufficient to alter the synthesis of the N-glycan chain in renal epithelial cells. We also show that ALG5 is required for PC1 maturation and membrane and ciliary localization and that heterozygous loss of ALG5 affects PC1 maturation. Overall, our results indicate that monoallelic variants of ALG5 lead to a disorder of the ADPKD-spectrum characterized by multiple small kidney cysts, progressive interstitial fibrosis, and kidney function decline.

Oxime Coupling of Active Site Inhibited Factor Seven with a Nonvolatile, Water-Soluble Fluorine-18 Labeled Aldehyde.

A nonvolatile fluorine-18 aldehyde prosthetic group was developed from [18F]SFB, and used for site-specific labeling of active site inhibited factor VII (FVIIai). FVIIai has a high affinity for tissue factor (TF), a transmembrane protein involved in angiogenesis, proliferation, cell migration, and survival of cancer cells. A hydroxylamine N-glycan modified FVIIai (FVIIai-ONH2) was used for oxime coupling with the aldehyde [18F]2 under mild and optimized conditions in an isolated RCY of 4.7 ± 0.9%, and a synthesis time of 267 ± 5 min (from EOB). Retained binding and specificity of the resulting [18F]FVIIai to TF was shown in vitro. TF-expression imaging capability was evaluated by in vivo PET/CT imaging in a pancreatic human xenograft cancer mouse model. The conjugate showed exceptional stability in plasma (>95% at 4 h) and a binding fraction of 90%. In vivo PET/CT imaging showed a mean tumor uptake of 3.8 ± 0.2% ID/g at 4 h post-injection, a comparable uptake in liver and kidneys, and low uptake in normal tissues. In conclusion, FVIIai was labeled with fluorine-18 at the N-glycan chain without affecting TF binding. In vitro specificity and a good in vivoimaging contrast at 4 h postinjection was demonstrated.

Reducing Macro- and Microheterogeneity of N-Glycans Enables the Crystal Structure of the Lectin and EGF-Like Domains of Human L-Selectin To Be Solved at 1.9 Å Resolution.

L-Selectin, a cell-adhesion receptor on the surface of most leukocytes, contains seven N-glycosylation sites. In order to obtain the crystal structure of human L-selectin, we expressed a shortened version of L-selectin comprising the C-type lectin and EGF-like domains (termed LE) and systematically analysed mutations of the three glycosylation sites (Asn22, Asn66 and Asn139) in order to reduce macroheterogeneity. After we further removed microheterogeneity, we obtained crystals that diffracted X-rays up to 1.9 Å from a variant (LE010) with exchanges N22Q and N139Q and one GlcNAc2 Man5 N-glycan chain attached to Asn66. Crystal-structure analysis showed that the terminal mannose of GlcNAc2 Man5 of one LE010 molecule was coordinated to Ca2+ in the binding site of a symmetry-related LE010. The orientation of the lectin and EGF-like domain was similar to the described "bent" conformation of E- and P-selectins. The Ca2+ -binding site reflects the binding mode seen in E- and P-selectin structures co-crystallised with ligands.

Evaluation of in vivo bioactivities of recombinant hypo- (FSH21/18) and fully- (FSH24) glycosylated human FSH glycoforms in Fshb null mice

The hormone - specific FSHβ subunit of the human FSH heterodimer consists of N-linked glycans at Asn7 and Asn24 residues that are co-translationally attached early during subunit biosynthesis. Differences in the number of N-glycans (none, one or two) on the human FSHβ subunit contribute to macroheterogeneity in the FSH heterodimer. The resulting FSH glycoforms are termed hypo-glycosylated (FSH21/18, missing either an Asn24 or Asn7 N-glycan chain on the β - subunit, respectively) or fully glycosylated (FSH24, possessing of both Asn7 and Asn24 N-linked glycans on the β - subunit) FSH. The recombinant versions of human FSH glycoforms (FSH21/18 and FSH24) have been purified and biochemically characterized. In vitro functional studies have indicated that FSH21/18 exhibits faster FSH- receptor binding kinetics and is much more active than FSH24 in every assay tested to date. However, the in vivo bioactivity of the hypo-glycosylated FSH glycoform has never been tested. Here, we evaluated the in vivo bioactivities of FSH glycoforms in Fshb null mice using a pharmacological rescue approach. In Fshb null female mice, both hypo- and fully-glycosylated FSH elicited an ovarian weight gain response by 48 h and induced ovarian genes in a dose- and time-dependent manner. Quantification by real time qPCR assays indicated that hypo-glycosylated FSH21/18 was bioactive in vivo and induced FSH-responsive ovarian genes similar to fully-glycosylated FSH24. Western blot analyses followed by densitometry of key signaling components downstream of the FSH-receptor confirmed that the hypo-glycosylated FSH21/18 elicited a response similar to that by fully-glycosylated FSH24 in ovaries of Fshb null mice. When injected into Fshb null males, hypo-glycosylated FSH21/18 was more active than the fully-glycosylated FSH24 in inducing FSH-responsive genes and Sertoli cell proliferation. Thus, our data establish that recombinant hypo-glycosylated human FSH21/18 glycoform elicits bioactivity in vivo similar to the fully-glycosylated FSH. Our studies may have clinical implications particularly in formulating FSH-based ovarian follicle induction protocols using a combination of different human FSH glycoforms.

Novel interactions of complex carbohydrates with peanut (PNA), Ricinus communis (RCA-I), Sambucus nigra (SNA-I) and wheat germ (WGA) agglutinins as revealed by the binding specificities of these lectins towards mucin core-2 O-linked and N-linked glycans and related structures.

Plant lectins through their multivalent quaternary structures bind intrinsically flexible oligosaccharides. They recognize fine structural differences in carbohydrates and interact with different sequences in mucin core 2 or complex-type N-glycan chain and also in healthy and malignant tissues. They are used in characterizing cellular and extracellular glycoconjugates modified in pathological processes. We study here, the complex carbohydrate-lectin interactions by determining the effects of substituents in mucin core 2 tetrasaccharide Galβ1-4GlcNAcβ1-6(Galβ1-3)GalNAcα-O-R and fetuin glycopeptides on their binding to agarose-immobilized lectins PNA, RCA-I, SNA-I and WGA. Briefly, in mucin core 2 tetrasaccharide (i) structures modified by α2-3/6-Sialyl LacNAc, LewisX and α1-3-Galactosyl LacNAc resulted in regular binding to PNA whereas compounds with 6-sulfo LacNAc displayed no-binding; (ii) strucures bearing α2-6-sialyl 6-sulfo LacNAc, or 6-sialyl LacdiNAc carbohydrates displayed strong binding to SNA-I; (iii) structures with α2-3/6-sialyl, α1-3Gal LacNAc or LewisX were non-binder to RCA-I and compounds with 6-sulfo LacNAc only displayed weak binding; (iv) structures containing LewisX, 6-Sulfo LewisX, α2-3/6-sialyl LacNAc, α2-3/6-sialyl 6-sulfo LacNAc and GalNAc Lewis-a were non-binding to WGA, those with α1-2Fucosyl, α1-3-Galactosyl LacNAc, α2-3-sialyl T-hapten plus 3'/6'sulfo LacNAc displayed weak binding, and compounds with α2-3-sialyl T-hapten, α2.6-Sialyl LacdiNAc, α2-3-sialyl D-Fucβ1-3 GalNAc and Fucα-1-2 D-Fucβ-1-3GalNAc displaying regular binding and GalNAc LewisX and LacdiNAc plus D-Fuc β-1-3 GalNAcα resulting in tight binding. RCA-I binds Fetuin triantennary asialoglycopeptide 100% after α-2-3 and 25% after α-2-6 sialylation, 30% after α-1-2 and 100% after α-1-3 fucosylation, and 50% after α-1-3 galactosylation. WGA binds 3-but not 6-Fucosyl chitobiose core. Thus, information on the influence of complex carbohydrate chain constituents on lectin binding is apparently essential for the potential application of lectins in glycoconjugate research.

Crystal structures of glycoside hydrolase family 3 β-glucosidase 1 from Aspergillus aculeatus

GH3 (glycoside hydrolase family 3) BGLs (β-glucosidases) from filamentous fungi have been widely and commercially used for the supplementation of cellulases. AaBGL1 (Aspergillus aculeatus BGL1) belongs to the GH3 and shows high activity towards cellooligosaccharides up to high degree of polymerization. In the present study we determined the crystal structure of AaBGL1. In addition to the substrate-free structure, the structures of complexes with glucose and various inhibitors were determined. The structure of AaBGL1 is highly glycosylated with 88 monosaccharides (18 N-glycan chains) in the dimer. The largest N-glycan chain comprises ten monosaccharides and is one of the largest glycans ever observed in protein crystal structures. A prominent insertion region exists in a fibronectin type III domain, and this region extends to cover a wide surface area of the enzyme. The subsite +1 of AaBGL1 is highly hydrophobic. Three aromatic residues are present at subsite +1 and are located in short loop regions that are uniquely present in this enzyme. There is a long cleft extending from subsite +1, which appears to be suitable for binding long cellooligosaccharides. The crystal structures of AaBGL1 from the present study provide an important structural basis for the technical improvement of enzymatic cellulosic biomass conversion.

Structural and mechanistic insight into N-glycan processing by endo-α-mannosidase

N-linked glycans play key roles in protein folding, stability, and function. Biosynthetic modification of N-linked glycans, within the endoplasmic reticulum, features sequential trimming and readornment steps. One unusual enzyme, endo-α-mannosidase, cleaves mannoside linkages internally within an N-linked glycan chain, short circuiting the classical N-glycan biosynthetic pathway. Here, using two bacterial orthologs, we present the first structural and mechanistic dissection of endo-α-mannosidase. Structures solved at resolutions 1.7-2.1 Å reveal a (β/α)(8) barrel fold in which the catalytic center is present in a long substrate-binding groove, consistent with cleavage within the N-glycan chain. Enzymatic cleavage of authentic Glc(1/3)Man(9)GlcNAc(2) yields Glc(1/3)-Man. Using the bespoke substrate α-Glc-1,3-α-Man fluoride, the enzyme was shown to act with retention of anomeric configuration. Complexes with the established endo-α-mannosidase inhibitor α-Glc-1,3-deoxymannonojirimycin and a newly developed inhibitor, α-Glc-1,3-isofagomine, and with the reducing-end product α-1,2-mannobiose structurally define the -2 to +2 subsites of the enzyme. These structural and mechanistic data provide a foundation upon which to develop new enzyme inhibitors targeting the hijacking of N-glycan synthesis in viral disease and cancer.

N-Glycosylation Is Critical for the Stability and Intracellular Trafficking of Glucose Transporter GLUT4

The facilitative glucose transporter GLUT4 plays a key role in regulating whole body glucose homeostasis. GLUT4 dramatically changes its distribution upon insulin stimulation, and insulin-resistant diabetes is often linked with compromised translocation of GLUT4 under insulin stimulation. To elucidate the functional significance of the sole N-glycan chain on GLUT4, wild-type GLUT4 and a GLUT4 glycosylation mutant conjugated with enhanced GFP were stably expressed in HeLa cells. The N-glycan contributed to the overall stability of newly synthesized GLUT4. Moreover, cell surface expression of wild-type GLUT4 in HeLa cells was elevated upon insulin treatment, whereas the glycosylation mutant lost the ability to respond to insulin. Subcellular distribution of the mutant was distinct from that of wild-type GLUT4, implying that the subcellular localization required for insulin-mediated translocation was impaired in the mutant protein. Interestingly, kifunensine-treated cells also lost sensitivity to insulin, suggesting the functional importance of the N-glycan structure for GLUT4 trafficking. The K(m) or turnover rates of wild-type and mutant GLUT4, however, were similar, suggesting that the N-glycan had little effect on transporter activity. These findings underscore the critical roles of the N-glycan chain in quality control as well as intracellular trafficking of GLUT4.

Approaching the secrets of N-glycosylation in Aspergillus fumigatus: characterization of the AfOch1 protein.

The mannosyltransferase Och1 is the key enzyme for synthesis of elaborated protein N-glycans in yeast. In filamentous fungi genes implicated in outer chain formation are present, but their function is unclear. In this study we have analyzed the Och1 protein of Aspergillus fumigatus. We provide first evidence that poly-mannosylated N-glycans exist in A. fumigatus and that their synthesis requires AfOch1 activity. This implies that AfOch1 plays a similar role as S. cerevisiae ScOch1 in the initiation of an N-glycan outer chain. A Δafoch1 mutant showed normal growth under standard and various stress conditions including elevated temperature, cell wall and oxidative stress. However, sporulation of this mutant was dramatically reduced in the presence of high calcium concentrations, suggesting that certain proteins engaged in sporulation require N-glycan outer chains to be fully functional. A characteristic feature of AfOch1 and Och1 homologues from other filamentous fungi is a signal peptide that clearly distinguishes them from their yeast counterparts. However, this difference does not appear to have consequences for its localization in the Golgi. Replacing the signal peptide of AfOch1 by a membrane anchor had no impact on its ability to complement the sporulation defect of the Δafoch1 strain. The mutant triggered a normal cytokine response in infected murine macrophages, arguing against a role of outer chains as relevant Aspergillus pathogen associated molecular patterns. Infection experiments provided no evidence for attenuation in virulence; in fact, according to our data the Δafoch1 mutant may even be slightly more virulent than the control strains.

Caenorhabditis elegans N-glycans containing a Gal-Fuc disaccharide unit linked to the innermost GlcNAc residue are recognized by C. elegans galectin LEC-6

We report a detailed structural analysis of the N-glycans of Caenorhabditis elegans recognized by C. elegans galectin LEC-6. Glycoproteins of C. elegans captured by an immobilized LEC-6 affinity adsorbent were isolated. The N-glycans of these glycoproteins were then liberated by hydrazinolysis and labeled with the fluorophore 2-aminopyridine (PA). The derived pyridylaminated (PA)-sugars were further fractionated by rechromatography on immobilized LEC-6 adsorbent and by reversed-phase high-performance liquid chromatography (HPLC). The structures of the PA-sugars thus obtained were analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS/MS) in conjunction with glycosidase digestion. We confirmed that all PA-sugars having affinity for LEC-6 contain a Gal-Fuc disaccharide unit, and that this unit is bound to the innermost GlcNAc residue of the N-glycan chain. The dissociation constants of LEC-6 for these glycans were measured by frontal affinity chromatography. LEC-6 exhibited higher affinity for oligosaccharides having a Gal-Fuc unit linked to position 6 of the innermost GlcNAc residue than for those having Galbeta1-4GlcNAc units. Affinity for the former disappeared, however, following treatment with beta-galactosidase. If the glycan contained a Hex-Fuc disaccharide linked to the penultimate GlcNAc residue, the affinity would be diminished. We propose, therefore, that the galectins of C. elegans utilize the Gal-Fuc disaccharide unit for recognition instead of the Gal-GlcNAc unit that is common in vertebrates.

N-glycans of core2 β(1,6)-N-acetylglucosaminyltransferase-I (C2GnT-I) but not those of α(1,3)-fucosyltransferase-VII (FucT-VII) are required for the synthesis of functional P-selectin glycoprotein ligand-1 (PSGL-1): effects on P-, L- and E-selectin binding

C2GnT-I [core2 beta(1,6)-N-acetyglucosaminyltransferase-I] and FucT-VII [alpha(1,3)-fucosyltransferase-VII] are the key enzymes for the biosynthesis of sialyl-Lewis x determinants on selectin ligands and therefore they represent good drug targets for the treatment of inflammatory disorders and other pathologies involving selectins. In the present study, we examined the importance of N-glycosylation for the ability of C2GnT-I and FucT-VII to generate functional selectin ligands, particularly the PSGL-1 (P-selectin glycoprotein ligand-1). We found that (i) both enzymes have their two N-glycosylation sites occupied, (ii) for C2GnT-I, the N-glycan chain linked to Asn-95 significantly contributes to the synthesis of functional PSGL-1 and is required to localize the enzyme to the cis/medial-Golgi compartment, (iii) all N-glycosylation-deficient proteins of FucT-VII displayr a dramatic impairment of their in vitro enzymatic activities, but retain their ability to fucosylate the core2-modified PSGL-I and to generate P- and L-selectin binding, and (iv) the glycomutants of FucT-VII fail to synthesize sialyl-Lewis x or to generate E-selectin binding unless core2-modified PSGL-1 is present. All combined, our results show a differential functional impact of N-glycosylation on C2GnT-1 and FucT-VII and disclose that a strongly reduced FucT-VII activity retains the ability to fucosylate PSGL-1 on the core2-based binding site(s) for the three selectins.

A simple, sensitive in vitro assay for cytoplasmic deglycosylation by peptide: N-glycanase

A cytoplasmic peptide: N-glycanase (PNGase) has been implicated in the proteasomal degradation of aberrant glycoproteins synthesized in the endoplasmic reticulum. The reaction is believed to be important for subsequent proteolysis by the proteasome since bulky N-glycan chains on misfolded glycoproteins may impair their efficient entry into the interior of the cylinder-shaped 20S proteasome, where its active site resides. This cytoplasmic enzyme was first detected in 1993 by a simple, sensitive assay method using 14C-labeled glycopeptide as a substrate. The deglycosylation reaction by PNGase brings about two major changes on substrate the peptide; one is removal of the N-glycan chain and the other is the introduction of a negative charge into the core peptide by converting the glycosylated asparagine residue(s) into an aspartic acid residue(s). The assay method we developed monitors these major changes in the core peptide, and the respective changes were detected by distinct analytical methods: i.e., paper chromatography and paper electrophoresis. This chapter will describe the simple, sensitive in vitro assay method for PNGase.

Suppression of cell death in primary rat hepatocytes by α 1-acid glycoprotein

In screening for effective additives for the long-term culture of hepatocytes, the hepatoprotective effect of α 1-acid glycoprotein (AGP) was observed. AGP prevented primary hepatocytes from undergoing cell death induced by the chemical toxin, bromobenzene. Moreover, AGP added to medium was found to maintain the number of viable hepatocytes for as long as 6 d. The hepatoprotective effect of AGP was lost by removing sialic acid groups at the N-glycan chain terminal of AGP. It is shown that the complete form of N-glycan chain is needed for the hepatoprotectivity of AGP.

Degradation of Corneodesmosome Proteins by Two Serine Proteases of the Kallikrein Family, SCTE/KLK5/hK5 and SCCE/KLK7/hK7

Corneodesmosin (CDSN), desmoglein 1 (DSG1), and desmocollin 1 (DSC1) are adhesive proteins of the extracellular part of the corneodesmosomes, the junctional structures that mediate corneocyte cohesion. The degradation of these proteins at the epidermis surface is necessary for desquamation. Two serine proteases of the kallikrein family synthesized as inactive precursors have been implicated in this process: the stratum corneum chymotryptic enzyme (SCCE/KLK7/hK7) and the stratum corneum tryptic enzyme (SCTE/KLK5/hK5). Here, we analyzed the capacity of these enzymes to cleave DSG1, DSC1, and epidermal or recombinant forms of CDSN, at an acidic pH close to that of the stratum corneum. SCCE directly cleaved CDSN and DSC1 but was unable to degrade DSG1. But incubation with SCTE induced degradation of the three corneodesmosomal components. Using the recombinant form of CDSN, either with its N-glycan chain or enzymatically deglycosylated, we also demonstrated that oligosaccharide residues do not protect CDSN against proteolysis by SCCE. Moreover, our results suggest that SCTE is able to activate the proform of SCCE. These results strongly suggest that the two kalikreins are involved in desquamation. A model is proposed for desquamation that could be regulated by a precisely controlled protease-protease inhibitor balance.

Glycosylation and over-expression of endometriosis-associated peritoneal haptoglobin.

Peritoneal endometriotic tissues synthesize and secrete haptoglobin (pHp), which has an analogous nucleotide sequence to hepatic haptoglobin found in serum (sHp). This study performed enzymatic digestions and lectin binding assays to determine if differences in protein glycosylation exist between sHp and pHp, which may provide insight into pHp function and/or identify epitopes for development of novel methods of medical management of endometriosis. To reduce the dependence on surgical collection of peritoneal tissues from women, recombinant peritoneal Hp (rpHp) was produced and its glycosylation analyzed for future functional studies. These results showed the apparent molecular weight of pHp was 3 kDa smaller than sHp. Desialylation and complete N-deglycosylation elicited similar shifts in sHp and pHp electrophoretic migration, suggesting similar sialic acid content and indicating the 3 kDa variance was due to carbohydrate content, not protein degradation, respectively. Sequential deglycosylation of the four sHp N-glycan chains caused a 3 kDa shift per N-glycan removed suggesting the 3 kDa difference between sHp and pHp may be one N-glycan chain. Lectin ELISA and lectin-blotting analyses demonstrated increased pHp and rpHp interactions with MAL and LTL but no difference in binding to SNL compared to sHp from healthy individuals, identifying variations in the ratios of alpha(2-3) to alpha(2-6) sialic acid and fucose residues. Recombinant pHp was 100-fold over-expressed with a similar glycosylation pattern to pHp, albeit in an unprocessed alpha-beta Hp polypeptide form. These results are the first to identify differences between pHp and sHp glycosylation and lay groundwork further studies to characterize anomalies in glycan composition and structure, which likely impart pHp with known immunomodulatory functions and may be used as epitopes for development of immune based therapeutics for novel, non-surgical management of endometriosis.