Glycobiology最新文献

Cytosolic peptide:N-glycanase (PNGase/NGLY1 in mammals), a widely conserved amidase in eukaryotes, catalyzes the removal of N-glycans from glycoproteins and contributes to the quality control system for nascent glycoproteins. Since the first report of a patient with an autosomal recessive genetic disorder caused by NGLY1 deficiency in 2012, over 150 cases have been identified globally. Among the potential biomarkers for NGLY1 deficiency, Asn-linked mono/oligosaccharides-Asn-GlcNAc and Asn-HexNAc-Hex-NeuAc-have emerged as the most consistently and markedly elevated molecules in the plasma or urine of affected patients. This study examined the Asn-GlcNAc biosynthetic pathway, demonstrating that cytosolic endo-β-N-acetylglucosaminidase (ENGase), the proteasome, and peptidases are essential for its generation. NGLY1-deficient models and patients exhibited accumulation of novel elongated forms of Asn-GlcNAc, including Asn-GlcNAc-GalNAc, Asn-GlcNAc-Gal, and Asn-GlcNAc-Gal-NeuAc, in cells, culture supernatant, plasma, and urine. Our findings indicate that Asn-GlcNAc and Asn-oligosaccharides (Asn-OSs) may serve as promising diagnostic tools for NGLY1 deficiency.

Heparan sulfate is present on the cell surface and within the extracellular matrix of most animal species, and it regulates various physiological processes by binding to a wide variety of functional proteins. The diverse array of sulfation patterns on the heparan sulfate chain enables specific binding to each protein. Of particular interest is the 3-O-sulfated (3S) structure of glucosamine residues, which is considered a key structure in determining binding specificity to functional proteins. However, 3-O-sulfation is a rare modification, which makes it difficult to analyze and hinders elucidation of its physiological function. Establishing a general method for measuring the 3S structure is thus important for continued progress in this research field. We previously developed an HPLC method to separate and quantify 13 components of heparan sulfate, including five 3S components, as a complete heparin lyase digestion product. Application of this method in routine analysis required the development of a standard sample for quantitation that is simple to prepare in large amounts and exhibits good stability. A heparan sulfate standard composed of 13 components in known quantities, designated HS13, was prepared for this purpose. A standard mixture of the 13 components can be obtained by digesting HS13 with a heparin lyase. A compositional analysis of heparan sulfate derived from various rat organs was then conducted to test the newly developed standard. The organ-specific distribution of each 3S component was elucidated, and it was confirmed that the 3S components in biological samples can be quantified by routine HPLC analysis.

The glycan distribution on cells is governed by the activities of different families of enzymes that are together called "glycoEnzymes." These include ~400 gene products or 2% of the proteome, that have recently been curated in an ontology called GlycoEnzOnto. With the goal of making this ontology more accessible to the larger biomedical and biotechnology community, we organized a web resource called GlycoEnzDB, presenting this enzyme classification in terms of enzyme function, the pathways that they participate in and their EC numbers. This information is linked to i) Figures from the "Essentials of Glycobiology" textbook, ii) General gene, enzyme and pathway data appearing in external databases, iii) Manual and generative-artificial intelligence (AI) based text describing the function and pathways regulated by these entities, iv) Single-cell expression data across cell lines, normal human cell-types and tissue, and v) CRISPR-knockout/activation/inactivation and Transcription factor activity predictions. Whereas these data are curated for human glycoEnzymes, the knowledge framework may be extended to other species also. The user-friendly web interface is accessible at www.virtualglycome.org/glycoenzdb.

Glycans found on the ErbB family of receptors (HER1, HER2, and HER3) represent promising targets for cancer treatment. Characterization and full quantification of the bivalent kinetic interactions of therapeutic antibodies against the ErbB family of receptors directly in their native cancer cellular environment represent a unique strategy to help overcome cancer drug resistance and to the development of more effective therapeutic drugs. In this study, surface plasmon resonance microscopy (SPRM) was implemented in a unique and innovative manner to quantify the bivalent kinetic interactions of monoclonal antibodies targeting HER1 (EFGR), HER2 and HER3 directly on whole BXPC3 pancreatic cancer cells under a glycosylated (native) and deglycosylated cellular environment. Results revealed in unprecedented detail that both the single-arm affinity and double-arm stronger avidity modes of binding interaction could be observed. For bivalent Cetuximab (anti-HER1) KDs of 151 nM and 4.6 nM were observed, for bivalent Herceptin (anti-HER2) KDs of 2 nM and 0.1 nM were observed, and for bivalent anti-HER3 KDs of 13 nM and 1.3 nM were observed. However, upon enzymatic N-deglycosylation of BXPC3 cells, HER1 and HER3 demonstrated significant increase in affinity of 1000-fold and 21-fold, respectively. In contrast, HER2 kinetic interactions were negligibly influenced by cellular N-deglycosylation of BXPC3 cells. This study highlights for the first time SPRM's unique ability to characterize the bivalent heterogeneous kinetic interactions of monoclonal antibodies with ErbB receptors on whole cancer cells, and to quantify the shielding influence of pancreatic cancer cell surface N-glycosylation on these interactions.

Human extracellular endosulfatases HSulf-1 and HSulf-2 catalyze the selective 6-O-desulfation of heparan sulfate (HS), critically shaping the sulfation code that governs glycosaminoglycan (GAG)-mediated signaling. A unique feature of these enzymes is their hydrophilic domain (HD), an intrinsically disordered, non-conserved segment absent from all other human sulfatases. Despite lacking homology to known protein domains, the HD has emerged as a key regulatory module for substrate recognition, enzyme localization, and processive activity along HS chains. In this review, we dissect the structural and functional roles of the HD, with emphasis on its dynamic interaction with HS motifs and potential modulation of protein-GAG complexes. We also explore how its intrinsically disordered nature may confer conformational flexibility advantageous for navigating the complex landscape of extracellular glycans. Given the implication of HSulfs in diverse physiological and pathological contexts, including cancer, the HD presents a promising therapeutic target for the selective inhibition of endosulfatase activity. We discuss the challenges and perspectives in targeting intrinsically disordered regions (IDRs) in GAG-binding proteins, and highlight how the HD of HSulfs provides a paradigmatic example of non-canonical domains orchestrating fine-tuned GAG editing in the extracellular matrix.

Carbohydrate-active enzymes are often associated in tandem with various functional and structural domains, including hydrolytic enzymes, carbohydrate-binding modules, lectin domains, domains of unknown function, signal peptides, and immunoglobulin folds. Among these, carbohydrate-binding modules and lectin domains are well known to influence carbohydrate-active enzyme activity. Here, we investigated the impact of naturally occurring tandem domains on the hydrolytic activity of two well-characterized multi-domain sialidases: Bifidobacterium bifidum sialidase (BbSia2) and Glaesserella parasuis sialidase (HpNanH). We found that the sialidase activity of the BbSia2 catalytic domain remained unaffected upon deletion of its naturally occurring tandem domains. In contrast, deleting the naturally occurring tandem domains of HpNanH significantly reduced its sialidase activity. We also found that the non-native tandem placement of a previously reported sialic acid-binding module, MU3, enhanced the sialidase activity of the HpNanH catalytic domain, with the C-terminal positioning of MU3 providing a greater enhancement. However, the non-native tandem placement of MU3 or the non-catalytic tandem domains of HpNanH failed to enhance the sialidase activity of the catalytic domain of another well-characterized sialidase - Clostridium perfringens sialidase (CpNanI). These results highlight the complex and context-dependent roles of tandem domains in modulating carbohydrate-active enzyme activity and have implications for enzyme engineering studies.

SULF1, a human extracellular heparan 6-O-endosulfatase isoform 1, plays a critical role in embryonic development and cancer progression by modulating the 6-O-sulfation of heparan sulfate proteoglycans. However, limited recombinant protein production has hindered structural and functional characterization. To address this issue, we optimized SULF1 expression in HEK293F and HEK293T cells. We achieved yields of 2.2 mg/L of culture media after Ni2+-affinity purification of greater than 80% purity, representing a substantial improvement compared to the reported expression systems. We demonstrated that co-expression of sulfatase-modifying factor 1 in this expression system is essential for enhancing SULF1 enzymatic activity, which depends on conversion of active site cysteine to Cα-formylglycine and the presence of a Ca2+ ion. We further showed that a marine fucosylated chondroitin sulfate polymer isolated from the sea cucumber Holothuria floridana inhibits SULF1 enzymatic activity with IC50 of 0.05 ± 0.006 μg/mL and 0.07 ± 0.008 μg/mL for the GlcNS6S-GlcA-GlcNS6S-IdoA2S-GlcNS6S-IdoA2S-GlcNS6S-GlcA and 4-methylumbelliferyl sulfate substrates, respectively. Kinetic analysis revealed a mixed-mode inhibition, characterized by alterations in Vmax at all inhibitor concentrations and Km at high inhibitor concentrations. Efficient SULF1 production also enabled us to develop specific monoclonal antibodies, which confirmed SULF1 expression in the stroma of head and neck squamous cell cancer tissues. Collectively, this study provides an efficient workflow for the production of active human SULF1, investigates SULF1 inhibitors, and characterizes anti-SULF1 monoclonal antibodies, which will support further studies of this enzyme in various pathophysiological conditions.