Glycan Extensions Research Articles

Xylosyltransferase Bump-and-hole Engineering to Chemically Manipulate Proteoglycans in Mammalian Cells.

Mammalian cells orchestrate signalling through interaction events on their surfaces. Proteoglycans are an intricate part of these interactions, carrying large glycosaminoglycan polysaccharides that recruit signalling molecules. Despite their importance in development, cancer and neurobiology, a relatively small number of proteoglycans have been identified. In addition to the complexity of glycan extension, biosynthetic redundancy in the first protein glycosylation step by two xylosyltransferase isoenzymes XT1 and XT2 complicates annotation of proteoglycans. Here, we develop a chemical genetic strategy that manipulates the glycan attachment site of cellular proteoglycans. By employing a tactic termed bump- and-hole engineering, we engineer the two isoenzymes XT1 and XT2 to specifically transfer a chemically modified xylose analogue to target proteins. The chemical modification contains a bioorthogonal tag, allowing the ability to visualise and profile target proteins modified by both transferases in mammalian cells. The versatility of our approach allows pinpointing glycosylation sites by tandem mass spectrometry, and exploiting the chemical handle to manufacture proteoglycans with defined GAG chains for cellular applications. Engineered XT enzymes permit a view into proteoglycan biology that is orthogonal to conventional techniques in biochemistry.

Deficient glycan extension and endoplasmic reticulum stresses in ALG3-CDG.

ALG3-CDG is a rare congenital disorder of glycosylation (CDG) with a clinical phenotype that includes neurological manifestations, transaminitis, and frequent infections. The ALG3 enzyme catalyzes the first step of endoplasmic reticulum (ER) luminal glycan extension by adding mannose from Dol-P-Man to Dol-PP-Man5GlcNAc2 (Man5) forming Dol-PP-Man6. Such glycan extension is the first and fastest cellular response to ER stress, which is deficient in ALG3-CDG. In this study, we provide evidence that the unfolded protein response (UPR) and ER-associated degradation activities are increased in ALG3-CDG patient-derived cultured skin fibroblasts and there is constitutive activation of UPR mediated by the IRE1-α pathway. In addition, we show that N-linked Man3-4 glycans are increased in cellular glycoproteins and secreted plasma glycoproteins with hepatic or non-hepatic origin. We found that like other CDGs such as ALG1- or PMM2-CDG, in transferrin, the assembling intermediate Man5 in ALG3-CDG, are likely further processed into a distinct glycan, NeuAc1Gal1GlcNAc1Man3GlcNAc2, probably by Golgi mannosidases and glycosyltransferases. We predict it to be a mono-antennary glycan with the same molecular weight as the truncated glycan described in MGAT2-CDG. In summary, this study elucidates multiple previously unrecognized biochemical consequences of the glycan extension deficiency in ALG3-CDG which will have important implications in the pathogenesis of CDG.

Deglycosylated RBD produced in Pichia pastoris as a low-cost sera COVID-19 diagnosis tool and a vaccine candidate.

During the COVID-19 outbreak, numerous tools including protein-based vaccines have been developed. The methylotrophic yeast Pichia pastoris (synonymous to Komagataella phaffii) is an eukaryotic cost-effective and scalable system for recombinant protein production, with the advantages of an efficient secretion system and the protein folding assistance of the secretory pathway of eukaryotic cells. In a previous work, we compared the expression of SARS-CoV-2 Spike Receptor Binding Domain in P. pastoris with that in human cells. Although the size and glycosylation pattern was different between them, their protein structural and conformational features were indistinguishable. Nevertheless, since high mannose glycan extensions in proteins expressed by yeast may be the cause of a nonspecific immune recognition, we deglycosylated RBD in native conditions. This resulted in a highly pure, homogenous, properly folded and monomeric stable protein. This was confirmed by circular dichroism and tryptophan fluorescence spectra and by SEC-HPLC, which were similar to those of RBD proteins produced in yeast or human cells. Deglycosylated RBD was obtained at high yields in a single step, and it was efficient in distinguishing between SARS-CoV-2-negative and positive sera from patients. Moreover, when the deglycosylated variant was used as an immunogen, it elicited a humoral immune response ten times greater than the glycosylated form, producing antibodies with enhanced neutralizing power and eliciting a more robust cellular response. The proposed approach may be used to produce at a low cost, many antigens that require glycosylation to fold and express, but do not require glycans for recognition purposes.

Altered expression of glycan patterns and glycan-related genes in the medial prefrontal cortex of the valproic acid rat model of autism.

Autism spectrum disorders (ASD) represent a group of neurodevelopmental defects characterized by social deficits and repetitive behaviors. Alteration in Glycosylation patterns could influence the nervous system development and contribute to the molecular mechanism of ASD. Interaction of environmental factors with susceptible genes may affect expressions of glycosylation-related genes and thus result in abnormal glycosylation patterns. Here, we used an environmental factor-induced model of autism by a single intraperitoneal injection of 400 mg/kg valproic acid (VPA) to female rats at day 12.5 post-conception. Following confirmation of reduced sociability and increased self-grooming behaviors in VPA-treated offspring, we analyzed the alterations in the expression profile of glycan patterns and glycan-related genes by lectin microarrays and RNA-seq, respectively. Lectin microarrays detected 14 significantly regulated lectins in VPA rats, with an up-regulation of high-mannose with antennary and down-regulation of Siaα2-3 Gal/GalNAc. Based on the KEGG and CAZy resources, we assembled a comprehensive list of 961 glycan-related genes to focus our analysis on specific genes. Of those, transcription results revealed that there were 107 differentially expressed glycan-related genes (DEGGs) after VPA treatment. Functional analysis of DEGGs encoding anabolic enzymes revealed that the process trimming to form core structure and glycan extension from core structure primarily changed, which is consistent with the changes in glycan patterns. In addition, the DEGGs encoding glycoconjugates were mainly related to extracellular matrix and axon guidance. This study provides insights into the underlying molecular mechanism of aberrant glycosylation after prenatal VPA exposure, which may serve as potential biomarkers for the autism diagnosis.

Chemoenzymatic Synthesis of Asymmetrically Branched Human Milk Oligosaccharide Lacto-N-Hexaose.

We herein reported the first chemoenzymatic synthesis of lacto-N-hexaose (LNH) by combining chemical carbohydrate synthesis with a selectively enzymatic glycosylation strategy. A tetrasaccharide core structure GlcNH2β1→3 (GlcNAcβ1→6) Galβ1→4Glc, a key precursor for subsequent enzymatic glycan extension toward asymmetrically branched human milk oligosaccharides, was synthesized in this work. When the order of galactosyltransferase-catalyzed reactions was appropriately arranged, the β1,4-galactosyl and β1,3-galactosyl moieties could be sequentially assembled on the C6-arm and C3-arm of the tetrasaccharide, respectively, to achieve an efficient LNH synthesis. Lacto-N-neotetraose (LNnH), another common human milk oligosaccharide, was also synthesized en route to the target LNH.

Expanding the phenotype, genotype and biochemical knowledge of ALG3-CDG.

Congenital disorders of glycosylation (CDGs) are a continuously expanding group of monogenic disorders of glycoprotein and glycolipid biosynthesis that cause multisystem diseases. Individuals with ALG3-CDG frequently exhibit severe neurological involvement (epilepsy, microcephaly, and hypotonia), ocular anomalies, dysmorphic features, skeletal anomalies, and feeding difficulties. We present 10 unreported individuals diagnosed with ALG3-CDG based on molecular and biochemical testing with 11 novel variants in ALG3, bringing the total to 40 reported individuals. In addition to the typical multisystem disease seen in ALG3-CDG, we expand the symptomatology of ALG3-CDG to now include endocrine abnormalities, neural tube defects, mild aortic root dilatation, immunodeficiency, and renal anomalies. N-glycan analyses of these individuals showed combined deficiencies of hybrid glycans and glycan extension beyond Man5 GlcNAc2 consistent with their truncated lipid-linked precursor oligosaccharides. This spectrum of N-glycan changes is unique to ALG3-CDG. These expanded features of ALG3-CDG facilitate diagnosis and suggest that optimal management should include baseline endocrine, renal, cardiac, and immunological evaluation at the time of diagnosis and with ongoing monitoring.

Comparison of human poly-N-acetyl-lactosamine synthase structure with GT-A fold glycosyltransferases supports a modular assembly of catalytic subsites

Poly-N-acetyl-lactosamine (poly-LacNAc) structures are composed of repeating [-Galβ(1,4)-GlcNAcβ(1,3)-]n glycan extensions. They are found on both N- and O-glycoproteins and glycolipids and play an important role in development, immune function, and human disease. The majority of mammalian poly-LacNAc is synthesized by the alternating iterative action of β1,3-N-acetylglucosaminyltransferase 2 (B3GNT2) and β1,4-galactosyltransferases. B3GNT2 is in the largest mammalian glycosyltransferase family, GT31, but little is known about the structure, substrate recognition, or catalysis by family members. Here we report the structures of human B3GNT2 in complex with UDP:Mg2+ and in complex with both UDP:Mg2+ and a glycan acceptor, lacto-N-neotetraose. The B3GNT2 structure conserves the GT-A fold and the DxD motif that coordinates a Mg2+ ion for binding the UDP-GlcNAc sugar donor. The acceptor complex shows interactions with only the terminal Galβ(1,4)-GlcNAcβ(1,3)- disaccharide unit, which likely explains the specificity for both N- and O-glycan acceptors. Modeling of the UDP-GlcNAc donor supports a direct displacement inverting catalytic mechanism. Comparative structural analysis indicates that nucleotide sugar donors for GT-A fold glycosyltransferases bind in similar positions and conformations without conserving interacting residues, even for enzymes that use the same donor substrate. In contrast, the B3GNT2 acceptor binding site is consistent with prior models suggesting that the evolution of acceptor specificity involves loops inserted into the stable GT-A fold. These observations support the hypothesis that GT-A fold glycosyltransferases employ coevolving donor, acceptor, and catalytic subsite modules as templates to achieve the complex diversity of glycan linkages in biological systems.

Structure and mechanism of the ER-based glucosyltransferase ALG6.

In eukaryotic protein N-glycosylation, a series of glycosyltransferases catalyse the biosynthesis of a dolichylpyrophosphate-linked oligosaccharide before its transfer onto acceptor proteins1. The final seven steps occur in the lumen of the endoplasmic reticulum (ER) and require dolichylphosphate-activated mannose and glucose as donor substrates2. The responsible enzymes-ALG3, ALG9, ALG12, ALG6, ALG8 and ALG10-are glycosyltransferases of the C-superfamily (GT-Cs), which are loosely defined as containing membrane-spanning helices and processing an isoprenoid-linked carbohydrate donor substrate3,4. Here we present the cryo-electron microscopy structure of yeast ALG6 at 3.0Å resolution, which reveals a previously undescribed transmembrane protein fold. Comparison with reported GT-C structures suggests that GT-C enzymes contain a modular architecture with a conserved module and a variable module, each with distinct functional roles. We used synthetic analogues of dolichylphosphate-linked and dolichylpyrophosphate-linked sugars and enzymatic glycan extension to generate donor and acceptor substrates using purified enzymes of the ALG pathway to recapitulate the activity of ALG6 in vitro. A second cryo-electron microscopy structure of ALG6 bound to an analogue of dolichylphosphate-glucose at 3.9Å resolution revealed the active site of the enzyme. Functional analysis of ALG6 variants identified a catalytic aspartate residue that probably acts as a general base. This residue is conserved in the GT-C superfamily. Our results define the architecture of ER-luminal GT-C enzymes and provide a structural basis for understanding their catalytic mechanisms.

Bypassing Phase Variation of Lipooligosaccharide (LOS): Using Heptose 1 Glycan Mutants To Establish Widespread Efficacy of Gonococcal Anti-LOS Monoclonal Antibody 2C7.

The sialylatable lacto-N-neotetraose (LNnT; Gal-GlcNAc-Gal-Glc) moiety from heptose I (HepI) of the lipooligosaccharide (LOS) of Neisseria gonorrhoeae undergoes positive selection during human infection. Lactose (Gal-Glc) from HepII, although phase variable, is commonly expressed in humans; loss of HepII lactose compromises gonococcal fitness in mice. Anti-LOS monoclonal antibody (MAb) 2C7, a promising antigonococcal immunotherapeutic that elicits complement-dependent bactericidal activity and attenuates gonococcal colonization in mice, recognizes an epitope comprised of lactoses expressed simultaneously from HepI and HepII. Glycan extensions beyond lactose on HepI modulate binding and function of MAb 2C7 in vitro Here, four gonococcal LOS mutants, each with lactose from HepII but fixed (unable to phase-vary) LOS HepI glycans extended beyond the lactose substitution of HepI (lactose alone, Gal-lactose, LNnT, or GalNAc-LNnT), were used to define how HepI glycan extensions affect (i) mouse vaginal colonization and (ii) efficacy in vitro and in vivo of a human IgG1 chimeric derivative of MAb 2C7 (2C7-Ximab) with a complement-enhancing E-to-G Fc mutation at position 430 (2C7-Ximab-E430G). About 10-fold lower 2C7-Ximab-E430G concentrations achieved similar complement-dependent killing of three gonococcal mutants with glycan extensions beyond lactose-substituted HepI (lactose alone, LNnT, or GalNAc-LNnT) as 2C7-Ximab (unmodified Fc). The fourth mutant (Gal-lactose) resisted direct complement-dependent killing but was killed approximately 70% by 2C7-Ximab-E430G in the presence of polymorphonuclear leukocytes and complement. Only mutants with (sialylatable) LNnT from HepI colonized mice for >3 days, reiterating the importance of LNnT sialylation for infection. 2C7-Ximab-E430G significantly attenuated colonization caused by the virulent mutants.

Presence of N-acetylgalactosamine/galactose residues on bronchioloalveolar cells during rat postnatal development.

In mammals, the alveolarization process develops predominantly after birth. Airway cells display a complex assemblage of glycans on their surface. These glycans, particularly terminal glycan extensions, are important effective carriers of information that change during the differentiation process. Nevertheless, few systematic data are reported about the cell surface sugar residue content during postnatal lung development. In the present work, we aimed to identify and semi-quantify N-acetylgalactosamine (GalNAc)/galactose (Gal) residues on the bronchioloalveolar cell surface in rat lung sections from 1-, 4-, 8- day old and adult animals and link these data with the lung glycocalyx composition. Horseradish peroxidase- conjugated lectin from Glycine max (soybean agglutinin, SBA) was used, and light microscopy methodologies were performed. SBA labelling intensity was studied before and after sialidase pre-treatment, in 1-, 4-, and 8-day-old animals and adult animals. For semi-quantitative evaluation of SBA binding intensity, two investigators performed the analysis independently, blinded to the type of experiment. Reactivity of the lectin was assessed in bronchiolar and respiratory portion/alveolar epithelial cell surfaces. We evidenced a stronger positive reaction when lung sections were pre-treated with neuraminidase before incubation with the lectin in 1- and 4-day-old animals and adult animals. These results were not so manifest in 8- day-old animals. This binding pattern, generally points towards the presence of terminal but mainly sub-terminal GalNAc/Gal residues probably capped by sialic acids on the rat bronchiolar/respiratory tract epithelial cells. As this glycan extension is common in Oand N-glycans, our results suggest that these glycan classes can be present in bronchioloalveolar cells immediately after birth and exist during the postnatal period. The results observed in eight-day-old rat lung sections may be due to the dramatic lung morphologic changes and the possible underlying biological mechanisms that occur during this age-moment.

The impact of aminopyrene trisulfonate (APTS) label in acceptor glycan substrates for profiling plant pectin β-galactosyltransferase activities

Aminopyrene trisulfonate (APTS)-labelled disaccharides are demonstrated to serve as readily accessible acceptor substrates for galactosyltransferase activities present in Arabidopsis microsome preparations. The reductive amination procedure used to install the fluorophore results in loss of the ring structure of the reducing terminal sugar unit, such that a single intact sugar ring is present, attached via an alditol tether to the aminopyrene fluorophore. The configuration of the alditol portion of the labelled acceptor, as well as the position of alditol galactosylation, substantially influence the ability of compounds to serve as Arabidopsis galactosyltransferase acceptor substrates. The APTS label exhibits an unexpected reaction-promoting effect that is not evident for structurally similar sulfonated aromatic fluorophores ANDS and ANTS. When APTS-labelled β-(1 → 4)-Gal3 was employed as an acceptor substrate with Arabidopsis microsomes, glycan extension generated β-(1 → 4)-galactan chains running to beyond 60 galactose residues. These studies demonstrate the potential of even very short glycan-APTS probes for assessing plant galactosyltransferase activities and the suitability CE-LIF for CAZyme profiling.

Phase-Variable Heptose I Glycan Extensions Modulate Efficacy of 2C7 Vaccine Antibody Directed against Neisseria gonorrhoeae Lipooligosaccharide.

Neisseria gonorrhoeae, the causative agent of the sexually transmitted infection, gonorrhea, has developed resistance to most conventional antibiotics. Safe and effective vaccines against gonorrhea are needed urgently. A candidate vaccine that targets a lipooligosaccharide (LOS) epitope recognized mAb 2C7 attenuates gonococcal burden in the mouse vaginal colonization model. Glycan extensions from the LOS core heptoses (HepI and HepII) are controlled by phase-variable LOS glycosyltransferase (lgt) genes; we sought to define how HepI glycan extensions affect mAb 2C7 function. Isogenic gonococcal mutants in which the lgt required for mAb 2C7 reactivity (lgtG) was genetically locked on and the lgt loci required for HepI variation (lgtA, lgtC, and lgtD) were genetically locked on or off in different combinations were created. We observed 100% complement-dependent killing by mAb 2C7 of a mutant that expressed lactose (Gal-Glc) from HepI, whereas a mutant that expressed Gal-Gal-Glc-HepI fully resisted killing (>100% survival). Mutants that elaborated 4- (Gal-GlcNAc-Gal-Glc-HepI) and 5-glycan (GalNAc-Gal-GlcNAc-Gal-Glc-HepI) structures displayed intermediate phenotypes (<50% killing with 2 μg/ml and >95% killing with 4 μg/ml mAb 2C7). The contrasting phenotypes of the lactose-HepI and the Gal-Gal-Glc-HepI LOS structures were recapitulated with phase variants of a recently isolated clinical strain. Despite lack of killing of the Gal-Gal-Glc-HepI mutants, mAb 2C7 deposited sufficient C3 on these bacteria for opsonophagocytic killing by human neutrophils. In conclusion, mAb 2C7 showed functional activity against all gonococcal HepI LOS structures defined by various lgtA/C/D on/off combinations, thereby providing further impetus for use of the 2C7 epitope in a gonococcal vaccine.

Regulation of Glycan Structures in Animal Tissues: TRANSCRIPT PROFILING OF GLYCAN-RELATED GENES

Glycan structures covalently attached to proteins and lipids play numerous roles in mammalian cells, including protein folding, targeting, recognition, and adhesion at the molecular or cellular level. Regulating the abundance of glycan structures on cellular glycoproteins and glycolipids is a complex process that depends on numerous factors. Most models for glycan regulation hypothesize that transcriptional control of the enzymes involved in glycan synthesis, modification, and catabolism determines glycan abundance and diversity. However, few broad-based studies have examined correlations between glycan structures and transcripts encoding the relevant biosynthetic and catabolic enzymes. Low transcript abundance for many glycan-related genes has hampered broad-based transcript profiling for comparison with glycan structural data. In an effort to facilitate comparison with glycan structural data and to identify the molecular basis of alterations in glycan structures, we have developed a medium-throughput quantitative real time reverse transcriptase-PCR platform for the analysis of transcripts encoding glycan-related enzymes and proteins in mouse tissues and cells. The method employs a comprehensive list of >700 genes, including enzymes involved in sugar-nucleotide biosynthesis, transporters, glycan extension, modification, recognition, catabolism, and numerous glycosylated core proteins. Comparison with parallel microarray analyses indicates a significantly greater sensitivity and dynamic range for our quantitative real time reverse transcriptase-PCR approach, particularly for the numerous low abundance glycan-related enzymes. Mapping of the genes and transcript levels to their respective biosynthetic pathway steps allowed a comparison with glycan structural data and provides support for a model where many, but not all, changes in glycan abundance result from alterations in transcript expression of corresponding biosynthetic enzymes.

Activity of recycling Golgi mannosyltransferases in the yeast endoplasmic reticulum.

In yeast primary N- and O-glycans are attached to proteins in the endoplasmic reticulum (ER), and they are elongated in the Golgi. Thus, glycan extension by Golgi enzymes has been taken as evidence for arrival of a protein in the Golgi. Two alpha 1,6-mannosyltransferase activity-containing multiprotein complexes have been reported to recycle between the Golgi and the ER, but since resident ER proteins are not Golgi-modified, Golgi enzymes were not thought to function in the ER. Here we show that when protein exit from the ER was blocked in COPII-defective yeast mutants, the N-glycans of vacuolar carboxypeptidase Y and a set of unidentified glycoproteins were decorated with an alpha 1,6-mannose residue, normally added in the Golgi by Och1p. Immunofluorescent staining demonstrated that Och1p accumulated in the ER under these conditions. Concomitantly, primary O-glycans of a secretory protein were extended, apparently by the medial Golgi transferase Mnt1p. Similar O-glycan extension occurred in wild-type cells when an HDEL-tagged protein was allowed to encounter glycosyltransferases in the Golgi during recycling between ER and Golgi. Golgi-specific glycosylation in the ER was reduced when Golgi-to-ER traffic was blocked, confirming that glycan extension in the ER was mainly due to recycling, rather than newly synthesized transferases.