Glycosylation on proteins of the intestine and perimicrovillar membrane of Triatoma (Meccus) pallidipennis,under different feeding conditions

Trypanosoma cruzi,the causative agent of Chagas disease, interacts with molecules in the midgut of its insect vector to multiply and reach the infective stage. Many studies suggest that the parasite binds to midgut-specific glycans. We identified several glycoproteins expressed in the intestine and perimicrovillar membrane (PMM) of Triatoma (Meccus) pallidipennis under different feeding conditions. In order to assess changes in protein-linked glycans, we performed lectin and immunoblot analyses on glycoprotein extracts from these intestinal tissues using well-characterized lectins, and an antibody, which collectively recognize a wide range of different glycans epitopes. We observed that the amount and composition of proteins and glycoproteins associated with different glycans structures changed over time in the intestines and PMM under different physiological conditions. PMM extracts contained a wide variety of glycoproteins with different sugar residues, including abundant high-mannose and complex sialylated glycans. We propose that these molecules could be involved in the process of parasite-vector interactions.     

Glycopeptide microarray for autoantibody detection in cancer

Evaluation of: Pedersen JW, Blixt O, Bennett EP et al. Seromic profiling of colorectal cancer patients with novel glycopeptide microarray. Int. J. Cancer 128(8), 1860–1871 (2011).

Autoantibodies to cancer-associated antigens hold promise as sensitive biomarkers for cancer detection. Based on this hypothesis, and knowing that O-glycans on proteins constitute a source of possible epitopes recognized by autoantibodies, Pedersen and colleagues have generated a glycopeptide array displaying a comprehensive library of glycopeptides and glycoproteins derived from human mucins. The profiling of sera immunoreactivity of colon cancer patients allowed the identification of cancer-associated autoantibodies to various mucin (MUC)1 and MUC4 glycopeptides carrying aberrant glycosylation. This article provides evidence for the value of glycopeptides displaying cancer-associated glycans in diagnostic applications, and opens new avenues for the expansion to other protein glycoforms, as well as to further applications of such a microarray strategy for other post-translational modifications of proteins in the search for cancer biomarker.     

A new method for detection of tumor driver‐dependent changes of protein sialylation in a colon cancer cell line reveals nectin‐3 as TGFBR2 target

Protein‐linked glycans play key roles in cell differentiation, cell–cell interactions, cell growth, adhesion and immune response. Aberrant glycosylation is a characteristic feature of tumor cells and is involved in tumor growth, escape from apoptosis, metastasis formation, and resistance to therapy. It can serve as cancer biomarker and treatment target. To enable comprehensive screening for the impact of tumor driving mutations in colorectal cancer cells we present a method for specific analysis of tumor driver‐induced glycome changes. The strategy is based on a combination of three technologies, that is recombinase‐mediated cassette exchange (RMCE), Click‐It chemistry and mass spectrometry. The new method is exemplified by the analysis of the impact of inactivating mutations of the TGF‐ß‐receptor type II (TGFBR2) on sialic acid incorporation into protein‐linked glycans of the colon cancer cell line HCT116. Overall, 70 proteins were found to show de novo sialic acid incorporation exclusively upon TGFBR2 expression whereas 7 proteins lost sialylation upon TGFBR2 reconstitution. Validation of detected candidate glycoproteins is demonstrated with the cell surface glycoprotein nectin‐3 known to be involved in metastasis, invasion and prognosis of various cancers. Altogether, our new approach can help to systematically puzzle out the influence of tumor‐specific mutations in a major signaling pathway, as exemplified by the TGFBR2 tumor suppressor, on the tumor glycome. It facilitates the identification of glycan‐based tumor markers that could be used for diagnostic and therapeutic applications. In principle the outlined strategy can be adapted to any cancer cell line, tumor driver mutation and several glycan‐building blocks.     

Strategies for Analysis of the Glycosylation of Proteins: Current Status and Future Perspectives

More than half of human proteins are glycosylated by a bewildering array of complex and heterogeneous N- and O-linked glycans. They function in myriad biological processes, including cell adhesion and signalling and influence the physical characteristics, stability, function, activity and immunogenicity of soluble glycoproteins. A single protein may be glycosylated differently to yield heterogenous glycoforms. Glycosylation analysis is of increasing interest in biomedical and biological research, the pharmaceutical and healthcare industry and biotechnology. This is because it is increasingly apparent that glycosylation changes in diseases, such as cancer, making it a promising target for development of clinically useful biomarkers and therapeutics. Furthermore, as the non-human cells employed in expression systems glycosylate their proteins very differently to human cells, and as glycosylation changes unpredictably under changing environmental conditions, glycans analysis for quality control, optimum efficacy and safety of recombinant glycoproteins destined for human therapeutic use is paramount. The complexities of carbohydrate chemistry make analysis challenging and while there are a variety of robust methodologies available for glycan analysis, there is currently a pressing need for the development of new, streamlined, high throughput approaches accessible to non-specialist laboratories.     

BGAL1 depletion boosts the level of β‐galactosylation of N‐ and O‐glycans in N. benthamiana

Glyco‐design of proteins is a powerful tool in fundamental studies of structure–function relationship and in obtaining profiles optimized for efficacy of therapeutic glycoproteins. Plants, particularly Nicotiana benthamiana, are attractive hosts to produce recombinant glycoproteins, and recent advances in glyco‐engineering facilitate customized N‐glycosylation of plant‐derived glycoproteins. However, with exception of monoclonal antibodies, homogenous human‐like β1,4‐galactosylation is very hard to achieve in recombinant glycoproteins. Despite significant efforts to optimize the expression of β1,4‐galactosyltransferase, many plant‐derived glycoproteins still exhibit incomplete processed N‐glycans with heterogeneous terminal galactosylation. The most obvious suspects to be involved in trimming terminal galactose residues are β‐galactosidases (BGALs) from the glycosyl hydrolase family GH35. To elucidate the so far uncharacterized mechanisms leading to the trimming of terminal galactose residues from glycans of secreted proteins, we studied a N. benthamiana BGAL known to be active in the apoplast (NbBGAL1). Here, we determined the NbBGAL1 subcellular localization, substrate specificity and in planta biological activity. We show that NbBGAL1 can remove β1,4‐ and β1,3‐galactose residues on both N‐ and O‐glycans. Transient BGAL1 down‐regulation by RNA interference (RNAi) and BGAL1 depletion by genome editing drastically reduce β‐galactosidase activity in N. benthamiana and increase the amounts of fully galactosylated complex N‐glycans on several plant‐produced glycoproteins. Altogether, our data demonstrate that NbBGAL1 acts on galactosylated complex N‐glycans of plant‐produced glycoproteins.     

Selective isolation and analysis of glycoprotein fractions and their glycomes from hepatocellular carcinoma sera

As one of the most important post‐translational modifications, the discovery, isolation, and identification of glycoproteins are becoming increasingly important. In this study, a Con A‐magnetic particle conjugate‐based method was utilized to selectively isolate the glycoproteins and their glycomes from the healthy donor and hepatocellular carcinoma (HCC) case sera. The isolated glycoproteins and their N‐linked glycans were identified by LC‐ESI‐MS/MS and MALDI‐TOF/TOF‐MS, respectively. A total of 93 glycoproteins from the healthy donors and 85 glycoproteins from the HCC cases were identified. There were 34 different glycoproteins shown between the healthy donors (21/34) and the HCC cases (13/34). Twenty‐eight glycans from the healthy donors and 30 glycans from the HCC cases were detected and there were 22 different glycans shown between the healthy donors (10/22) and HCC cases (12/22). Among these glycoproteins, 50 were known to be N‐linked glycoproteins and three novel glycopeptides from two predicted potential glycoproteins were discovered. Moreover, lectin blotting, Western blotting and lectin/glyco‐antibody microarrays were applied to definitely elucidate the change of selective protein expressions and their glycosylation levels, the results indicated that the differences of the identified glycoproteins between the healthy donors and HCC cases were caused by the change of both protein expression and their glycosylation levels.     

Wisteria floribunda agglutinin positive glycobiomarkers: a unique lectin as a serum biomarker probe in various diseases

Introduction: Serum proteins are generally glycosylated and solubilized, and are thus present as glycoproteins. The glycan structure of glycoproteins reflects cell differentiation status; glycan structures generated by diseased cells are distinguishable from those produced by healthy cells. Proteins may therefore serve as markers of tissues that secrete them. Several strategies for the identification of novel serum biomarkers using a combination of glycoscience-based technologies have been recently proposed. The selection of lectins for use as probes for identification of altered glycan structures represents a critical step.

Areas covered: This review describes the identification of Wisteria floribunda agglutinin (WFA) as a probe that recognizes the altered glycan structure of glycoproteins secreted by diseased cells. WFA may be employed as a probe for several diseases, e.g., liver fibrosis, liver cirrhosis, prostate cancer, ovarian cancer, and IgA nephropathy. The advantage of employing WFA as a serum biomarker probe is that only very small amounts of WFA-positive glycoproteins are present in serum; therefore, WFA background in serum is very low.

Expert commentary: Based on the findings to date, several WFA-positive serum biomarkers may be measured without pre-purification of target glycoproteins, indicating their utility as serum biomarkers in patients with various diseases.     

Identification of N-glycosylated proteins from the central nervous system of Drosophila melanogaster

Although the function of many glycoproteins in the nervous system of fruit flies is well understood, information about the glycosylation profile and glycan attachment sites for such proteins is scarce. In order to fill this gap and to facilitate the analysis of N-linked glycosylation in the nervous system, we have performed an extensive survey of membrane-associated glycoproteins and their N-glycosylation sites isolated from the adult Drosophila brain. Following subcellular fractionation and trypsin digestion, we used different lectin affinity chromatography steps to isolate N-glycosylated glycopeptides. We identified a total of 205 glycoproteins carrying N-linked glycans and revealed their 307 N-glycan attachment sites. The size of the resulting dataset furthermore allowed the statistical characterization of amino acid distribution around the N-linked glycosylation sites. Glycan profiles were analyzed separately for glycopeptides that were strongly and weakly bound to Concanavalin A (Con A), or that failed to bind Concanavalin A, but did bind to wheat germ agglutinin (WGA). High- or paucimannosidic glycans dominated each of the profiles, although the wheat germ agglutinin-bound glycan population was enriched in more extensively processed structures. A sialylated glycan structure was unambiguously detected in the wheat germ agglutinin-bound fraction. Despite the large amount of starting material, insufficient amount of glycopeptides was retained by the Wisteria floribunda (WFA) and Sambucus nigra columns to allow glycan or glycoprotein identification, providing further evidence that the vast majority of glycoproteins in the adult Drosophila brain carry primarily high-mannose, paucimannose, and hybrid glycans. The obtained results should facilitate future genetic and molecular approaches addressing the role of N-glycosylation in the central nervous system (CNS) of Drosophila.     

Automated measurement of site‐specific N‐glycosylation occupancy with SWATH‐MS

Asparagine‐linked glycosylation is a common post‐translational modification of proteins catalyzed by oligosaccharyltransferase that is important in regulating many aspects of protein function. Analysis of protein glycosylation, including glycoproteomic measurement of the site‐specific extent of glycosylation, remains challenging. Here, we developed methods combining enzymatic deglycosylation and protease digestion with SWATH‐MS to enable automated measurement of site‐specific occupancy at many glycosylation sites. Deglycosylation with peptide‐endoglycosidase H, leaving a remnant N‐acetylglucosamine on asparagines previously carrying high‐mannose glycans, followed by trypsin digestion allowed robust automated measurement of occupancy at many sites. Combining deglycosylation with the more general peptide‐N‐glycosidase F enzyme with AspN protease digest allowed robust automated differentiation of nonglycosylated and deglycosylated forms of a given glycosylation site. Ratiometric analysis of deglycosylated peptides and the total intensities of all peptides from the corresponding proteins allowed relative quantification of site‐specific glycosylation occupancy between yeast strains with various isoforms of oligosaccharyltransferase. This approach also allowed robust measurement of glycosylation sites in human salivary glycoproteins. This method for automated relative quantification of site‐specific glycosylation occupancy will be a useful tool for research with model systems and clinical samples.     

High throughput screening for compounds that alter muscle cell glycosylation identifies new role for N-glycans in regulating sarcolemmal protein abundance and laminin binding

Duchenne muscular dystrophy is an X-linked disorder characterized by loss of dystrophin, a cytoskeletal protein that connects the actin cytoskeleton in skeletal muscle cells to extracellular matrix. Dystrophin binds to the cytoplasmic domain of the transmembrane glycoprotein β-dystroglycan (β-DG), which associates with cell surface α-dystroglycan (α-DG) that binds laminin in the extracellular matrix. β-DG can also associate with utrophin, and this differential association correlates with specific glycosylation changes on α-DG. Genetic modification of α-DG glycosylation can promote utrophin binding and rescue dystrophic phenotypes in mouse dystrophy models. We used high throughput screening with the plant lectin Wisteria floribunda agglutinin (WFA) to identify compounds that altered muscle cell surface glycosylation, with the goal of finding compounds that increase abundance of α-DG and associated sarcolemmal glycoproteins, increase utrophin usage, and increase laminin binding. We identified one compound, lobeline, from the Prestwick library of Food and Drug Administration-approved compounds that fulfilled these criteria, increasing WFA binding to C2C12 cells and to primary muscle cells from wild type and mdx mice. WFA binding and enhancement by lobeline required complex N-glycans but not O-mannose glycans that bind laminin. However, inhibiting complex N-glycan processing reduced laminin binding to muscle cell glycoproteins, although O-mannosylation was intact. Glycan analysis demonstrated a general increase in N-glycans on lobeline-treated cells rather than specific alterations in cell surface glycosylation, consistent with increased abundance of multiple sarcolemmal glycoproteins. This demonstrates the feasibility of high throughput screening with plant lectins to identify compounds that alter muscle cell glycosylation and identifies a novel role for N-glycans in regulating muscle cell function.     

Glycopeptide microarray for autoantibody detection in cancer

Evaluation of: Pedersen JW, Blixt O, Bennett EP et al. Seromic profiling of colorectal cancer patients with novel glycopeptide microarray. Int. J. Cancer 128(8), 1860-1871 (2011). Autoantibodies to cancer-associated antigens hold promise as sensitive biomarkers for cancer detection. Based on this hypothesis, and knowing that O-glycans on proteins constitute a source of possible epitopes recognized by autoantibodies, Pedersen and colleagues have generated a glycopeptide array displaying a comprehensive library of glycopeptides and glycoproteins derived from human mucins. The profiling of sera immunoreactivity of colon cancer patients allowed the identification of cancer-associated autoantibodies to various mucin (MUC)1 and MUC4 glycopeptides carrying aberrant glycosylation. This article provides evidence for the value of glycopeptides displaying cancer-associated glycans in diagnostic applications, and opens new avenues for the expansion to other protein glycoforms, as well as to further applications of such a microarray strategy for other post-translational modifications of proteins in the search for cancer biomarker.     

Identification of Plasma Membrane Glycoproteins Specific to Human Glioblastoma Multiforme Cells Using Lectin Arrays and LC‐MS/MS

Glioblastoma, also known as glioblastoma multiforme (GBM), is the most malignant type of brain cancer and has poor prognosis with a median survival of less than one year. While the structural changes of tumor cell surface carbohydrates are known to be associated with invasive behavior of tumor cells, the cell surface glycoproteins to differentiate the low‐ and high‐grade glioma cells can be potential diagnostic markers and therapeutic targets for GBMs. In the present study, lectin arrays consisting of eight lectins were employed to explore cell surface carbohydrate expression patterns on low‐grade oligodendroglioma cells (Hs683) and GBM cells (T98G). Griffonia simplicifolia I (GS I) was found to selectively bind to T98G cells and not to Hs683 cells. For identification of the glioblastoma‐specific cell surface markers, the glycoproteins from each cell type were captured by a GS I lectin column and analyzed by LC‐MS/MS. The identified proteins from the two cell types were quantified using label‐free quantitative analysis based on spectral counting. Of cell surface glycoproteins showing significant increases in T98G cells, five proteins were selected for verification of both protein and glycosylation level changes using Western blot and GS I lectin‐based immunosorbent assay.     

Carbohydrate‐binding agents act as potent trypanocidals that elicit modifications in VSG glycosylation and reduced virulence in Trypanosoma brucei

The surface of Trypanosoma brucei is covered by a dense coat of glycosylphosphatidylinositol‐anchored glycoproteins. The major component is the variant surface glycoprotein (VSG) which is glycosylated by both paucimannose and oligomannose N‐glycans. Surface glycans are poorly accessible and killing mediated by peptide lectin–VSG complexes is hindered by active endocytosis. However, contrary to previous observations, here we show that high‐affinity carbohydrate binding agents bind to surface glycoproteins and abrogate growth of T. brucei bloodstream forms. Specifically, binding of the mannose‐specific Hippeastrum hybrid agglutinin (HHA) resulted in profound perturbations in endocytosis and parasite lysis. Prolonged exposure to HHA led to the loss of triantennary oligomannose structures in surface glycoproteins as a result of genetic rearrangements that abolished expression of the oligosaccharyltransferase TbSTT3B gene and yielded novel chimeric enzymes. Mutant parasites exhibited markedly reduced infectivity thus demonstrating the importance of specific glycosylation patterns in parasite virulence.     

MALDI Mass Spectrometry Imaging of Early‐ and Late‐Stage Serous Ovarian Cancer Tissue Reveals Stage‐Specific N‐Glycans

Epithelial ovarian cancer is one of the most fatal gynecological malignancies in adult women. As studies on protein N‐glycosylation have extensively reported aberrant patterns in the ovarian cancer tumor microenvironment, obtaining spatial information will uncover tumor‐specific N‐glycan alterations in ovarian cancer development and progression. matrix‐assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) is employed to investigate N‐glycan distribution on formalin‐fixed paraffin‐embedded ovarian cancer tissue sections from early‐ and late‐stage patients. Tumor‐specific N‐glycans are identified and structurally characterized by porous graphitized carbon‐liquid chromatography‐electrospray ionization‐tandem mass spectrometry (PGC‐LC‐ESI‐MS/MS), and then assigned to high‐resolution images obtained from MALDI‐MSI. Spatial distribution of 14 N‐glycans is obtained by MALDI‐MSI and 42 N‐glycans (including structural and compositional isomers) identified and structurally characterized by LC‐MS. The spatial distribution of oligomannose, complex neutral, bisecting, and sialylated N‐glycan families are localized to the tumor regions of late‐stage ovarian cancer patients relative to early‐stage patients. Potential N‐glycan diagnostic markers that emerge include the oligomannose structure, (Hex)₆ + (Man)₃(GlcNAc)₂, and the complex neutral structure, (Hex)₂ (HexNAc)₂ (Deoxyhexose)₁ + (Man)₃(GlcNAc)₂. The distribution of these markers is evaluated using a tissue microarray of early‐ and late‐stage patients.     

Identification of unique glycoisoforms of vitamin D-binding protein and haptoglobin as biomarker candidates in hepatocarcinogenesis of STAM mice

Hepatocellular carcinoma (HCC) is the major subtype of primary liver cancer, and is typically diagnosed late in its course. Considering the limitations and the reluctance of patients to undergo a liver biopsy, a reliable, noninvasive diagnostic marker that predicts and assesses the treatment and prognosis of HCC is needed. With recent technological advances of mass spectrometry, glycomics is gathering momentum and holds substantial potential to discover new glycan markers in cancer research. Here, to discover specific glycan markers for the early diagnosis of HCC, we analyzed the glycan profiles of gel-separated serum proteins of progressive liver disease model mice. By focused protein glycomics of 12 gel-separated glycoproteins using sera from the mouse models, we revealed the entire profile of glycans in each major serum protein. We found that the levels of trisialylated triantennary glycans of haptoglobin and vitamin D-binding protein increased significantly as the disease progressed, while the alteration in these protein levels were modest. Furthermore, these glycan increases were not observed in age-matched control mice. In conclusion, our approach has identified specific glycan marker candidates for the early diagnosis of HCC.     

Altered glycosylation in cancer: A promising target for biomarkers and therapeutics

Glycosylation is a well-regulated cell and microenvironment specific post-translational modification. Several glycosyltransferases and glycosidases orchestrate the addition of defined glycan structures on the proteins and lipids. Recent advances and systemic approaches in glycomics have significantly contributed to a better understanding of instrumental roles of glycans in health and diseases. Emerging research evidence recognized aberrantly glycosylated proteins as the modulators of the malignant phenotype of cancer cells. The Cancer Genome Atlas has identified alterations in the expressions of glycosylation-specific genes that are correlated with cancer progression. However, the mechanistic basis remains poorly explored. Recent researches have shown that specific changes in the glycan structures are associated with 'stemness' and epithelial-to-mesenchymal transition of cancer cells. Moreover, epigenetic changes in the glycosylation pattern make the tumor cells capable of escaping immunosurveillance mechanisms. The deciphering roles of glycans in cancer emphasize that glycans can serve as a source for the development of novel clinical biomarkers. The ability of glycans in intervening various stages of tumor progression and the biosynthetic pathways involved in glycan structures constitute a promising target for cancer therapy. Advances in the knowledge of innovative strategies for identifying the mechanisms of glycan-binding proteins are hoped to hold great potential in cancer therapy. This review discusses the fundamental role of glycans in regulating tumorigenesis and tumor progression and provides insights into the influence of glycans in the current tactics of targeted therapies in the clinical setting.     

Deletion of fucose residues in plant N‐glycans by repression of the GDP‐mannose 4,6‐dehydratase gene using virus‐induced gene silencing and RNA interference

Production of pharmaceutical glycoproteins in plants has many advantages in terms of safety and reduced costs. However, plant‐produced glycoproteins have N‐glycans with plant‐specific sugar residues (core β‐1,2‐xylose and α‐1,3‐fucose) and a Lewis a (Le^a) epitope, i.e., Galβ(1‐3)[Fucα(1‐4)]GlcNAc. Because these sugar residues and glycan structures seemed to be immunogenic, several attempts have been made to delete them by repressing their respective glycosyltransferase genes. However, until date, such deletions have not been successful in completely eliminating the fucose residues. In this study, we simultaneously reduced the plant‐specific core α‐1,3‐fucose and α‐1,4‐fucose residues in the Le^a epitopes by repressing the Guanosine 5′‐diphosphate (GDP)‐D‐mannose 4,6‐dehydratase (GMD) gene, which is associated with GDP‐L‐fucose biosynthesis, in Nicotiana benthamiana plants. Repression of GMD was achieved using virus‐induced gene silencing (VIGS) and RNA interference (RNAi). The proportion of fucose‐free N‐glycans found in total soluble protein from GMD gene‐repressed plants increased by 80% and 95% following VIGS and RNAi, respectively, compared to wild‐type plants. A small amount of putative galactose substitution in N‐glycans from the NbGMD gene‐repressed plants was observed, similar to what has been previously reported GMD‐knockout Arabidopsis mutant. On the other hand, the recombinant mouse granulocyte‐macrophage colony‐stimulating factor (GM‐CSF) with fucose‐deleted N‐glycans was successfully produced in NbGMD‐RNAi transgenic N. benthamiana plants. Thus, repression of the GMD gene is thus very useful for deleting immunogenic total fucose residues and facilitating the production of pharmaceutical glycoproteins in plants.     

Glycoproteomic analyses of ovarian cancer cell lines and sera from ovarian cancer patients show distinct glycosylation changes in individual proteins

Ovarian cancer is difficult to diagnose in women because symptoms of the disease are often not noticed until the disease has progressed to an advanced untreatable stage. Although a serum test, CA125, is currently available to assist with monitoring treatment of ovarian cancer, this test lacks the necessary specificity and sensitivity for early detection. Therefore, better biomarkers of ovarian cancer are needed. A glycoprotein analysis approach was undertaken using high resolution Fourier transform ion cyclotron resonance mass spectrometry to analyze glycosylated proteins present in the conditioned media of ovarian cancer cell lines and in sera obtained from ovarian cancer patients and normal controls. In this study, glycosylated proteins were separated by gel electrophoresis, and individual glycoproteins were selected for glycosylation analysis and protein identification. The attached glycans from each protein were released and profiled by mass spectrometry. Glycosylation of a mucin protein and a large glycosylated protein isolated from the ES2 ovarian cancer cell line was determined to consist of mostly O-linked glycans. Four prominent glycoproteins of approximate 517, 370, 250, 163 kDa from serum samples were identified as two forms of apolipoprotein B-100, fibronectin, and immunoglobulin A1, respectively. Mass spectrometric analysis of glycans isolated from apolipoprotein B-100 (517 kD) showed the presence of small, specific O-linked oligosaccharides. In contrast, analysis of fibronectin (250 kD) and immunoglobulin A1 (163 kD) produced N-linked glycan fragments in forms that were sufficiently different from the glycans obtained from the corresponding protein band present in the normal serum samples. This study shows that not only a single protein but several are aberrantly glycosylated, and those abnormal glycosylation changes can be detected and may ultimately serve as glycan biomarkers for ovarian cancer.     

Human protein paucimannosylation: cues from the eukaryotic kingdoms

Paucimannosidic proteins (PMPs) are bioactive glycoproteins carrying truncated α‐ or β‐mannosyl‐terminating asparagine (N)‐linked glycans widely reported across the eukaryotic domain. Our understanding of human PMPs remains limited, despite findings documenting their existence and association with human disease glycobiology. This review comprehensively surveys the structures, biosynthetic routes and functions of PMPs across the eukaryotic kingdoms with the aim of synthesising an improved understanding on the role of protein paucimannosylation in human health and diseases. Convincing biochemical, glycoanalytical and biological data detail a vast structural heterogeneity and fascinating tissue‐ and subcellular‐specific expression of PMPs within invertebrates and plants, often comprising multi‐α1,3/6‐fucosylation and β1,2‐xylosylation amongst other glycan modifications and non‐glycan substitutions e.g. O‐methylation. Vertebrates and protists express less‐heterogeneous PMPs typically only comprising variable core fucosylation of bi‐ and trimannosylchitobiose core glycans. In particular, the Manα1,6Manβ1,4GlcNAc(α1,6Fuc)β1,4GlcNAcβAsn glycan (M2F) decorates various human neutrophil proteins reportedly displaying bioactivity and structural integrity demonstrating that they are not degradation products. Less‐truncated paucimannosidic glycans (e.g. M3F) are characteristic glycosylation features of proteins expressed by human cancer and stem cells. Concertedly, these observations suggest the involvement of human PMPs in processes related to innate immunity, tumorigenesis and cellular differentiation. The absence of human PMPs in diverse bodily fluids studied under many (patho)physiological conditions suggests extravascular residence and points to localised functions of PMPs in peripheral tissues. Absence of PMPs in Fungi indicates that paucimannosylation is common, but not universally conserved, in eukaryotes. Relative to human PMPs, the expression of PMPs in plants, invertebrates and protists is more tissue‐wide and constitutive yet, similar to their human counterparts, PMP expression remains regulated by the physiology of the producing organism and PMPs evidently serve essential functions in development, cell–cell communication and host–pathogen/symbiont interactions. In most PMP‐producing organisms, including humans, the N‐acetyl‐β‐hexosaminidase isoenzymes and linkage‐specific α‐mannosidases are glycoside hydrolases critical for generating PMPs via N‐acetylglucosaminyltransferase I (GnT‐I)‐dependent and GnT‐I‐independent truncation pathways. However, the identity and structure of many species‐specific PMPs in eukaryotes, their biosynthetic routes, strong tissue‐ and development‐specific expression, and diverse functions are still elusive. Deep exploration of these PMP features involving, for example, the characterisation of endogenous PMP‐recognising lectins across a variety of healthy and N‐acetyl‐β‐hexosaminidase‐deficient human tissue types and identification of microbial adhesins reactive to human PMPs, are amongst the many tasks required for enhanced insight into the glycobiology of human PMPs. In conclusion, the literature supports the notion that PMPs are significant, yet still heavily under‐studied biomolecules in human glycobiology that serve essential functions and create structural heterogeneity not dissimilar to other human N‐glycoprotein types. Human PMPs should therefore be recognised as bioactive glycoproteins that are distinctly different from the canonical N‐glycoprotein classes and which warrant a more dedicated focus in glycobiological research.