综述: 卵巢癌生物标志物的糖链谱研究进展

糖类抗原125(CA125)被认为是卵巢癌诊断的“金标准”,但在临床应用中普遍存在着特异性不高的问题．肿瘤形成和发展过程中常伴有糖基化修饰异常和糖链结构的改变,不同的肿瘤具有特异的异常糖链结构．近年来,借助凝集素芯片、多重质谱分析等糖蛋白组学和糖组学研究技术,发现不同来源CA125的O-糖链和N-糖链结构存在着明显的微观不均一性,以这些特征性糖链结构为标志物,可以显著提高CA125对卵巢癌的诊断特异性．在过去的10年,研究者们除对CA125糖链结构和糖基化模式做了深入的研究外,还利用糖组的研究方法,直接对来自卵巢癌患者血液、体液(腹水、囊泡液等)中糖蛋白的糖链做了精细的结构解析,结果显示,可有效鉴别卵巢癌患者和健康志愿者的特异性N-糖链结构,有可能成为灵敏度高和特异性好的卵巢癌生物标志物．卵巢癌生物标志物研究发展的总趋势是从传统的对蛋白质的定性和定量研究,逐步转向于对标志物糖基化修饰和特异性糖链结构的鉴定以及定量分析．本文从糖组学的视角,对卵巢癌标志物糖组学的研究现状及发展趋势进行了综述和展望．     

Focused differential glycan analysis with the platform antibody-assisted lectin profiling for glycan-related biomarker verification

Protein glycosylation is a critical subject attracting increasing attention in the field of proteomics as it is expected to play a key role in the investigation of histological and diagnostic biomarkers. In this context, an enormous number of glycoproteins have now been nominated as disease-related biomarkers. However, there is no appropriate strategy in the current proteome platform to qualify such marker candidate molecules, which relates their specific expression to particular diseases. Here, we present a new practical system for focused differential glycan analysis in terms of antibody-assisted lectin profiling (ALP). In the developed procedure, (i) a target protein is enriched from clinic samples (e.g. tissue extracts, cell supernatants, or sera) by immunoprecipitation with a specific antibody recognizing a core protein moiety; (ii) the target glycoprotein is quantified by immunoblotting using the same antibody used in (i); and (iii) glycosylation difference is analyzed by means of antibody-overlay lectin microarray, an application technique of an emerging glycan profiling microarray. As model glycoproteins having either N-linked or O-linked glycans, prostate-specific antigen or podoplanin, respectively, were subjected to systematic ALP analysis. As a result, specific signals corresponding to the target glycoprotein glycans were obtained at a sub-picomole level with the aid of specific antibodies, whereby disease-specific or tissue-specific glycosylation changes could be observed in a rapid, reproducible, and high-throughput manner. Thus, the established system should provide a powerful pipeline in support of on-going efforts in glyco-biomarker discovery.     

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.     

Multi‐scale spatial heterogeneity of pectic rhamnogalacturonan I (RG–I) structural features in tobacco seed endosperm cell walls

Plant cell walls are complex configurations of polysaccharides that fulfil a diversity of roles during plant growth and development. They also provide sets of biomaterials that are widely exploited in food, fibre and fuel applications. The pectic polysaccharides, which comprise approximately a third of primary cell walls, form complex supramolecular structures with distinct glycan domains. Rhamnogalacturonan I (RG–I) is a highly structurally heterogeneous branched glycan domain within the pectic supramolecule that contains rhamnogalacturonan, arabinan and galactan as structural elements. Heterogeneous RG–I polymers are implicated in generating the mechanical properties of cell walls during cell development and plant growth, but are poorly understood in architectural, biochemical and functional terms. Using specific monoclonal antibodies to the three major RG–I structural elements (arabinan, galactan and the rhamnogalacturonan backbone) for in situ analyses and chromatographic detection analyses, the relative occurrences of RG–I structures were studied within a single tissue: the tobacco seed endosperm. The analyses indicate that the features of the RG–I polymer display spatial heterogeneity at the level of the tissue and the level of single cell walls, and also heterogeneity at the biochemical level. This work has implications for understanding RG–I glycan complexity in the context of cell‐wall architectures and in relation to cell‐wall functions in cell and tissue development.     

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.     

Comparative profiling of serum glycoproteome by sequential purification of glycoproteins and 2-nitrobenzensulfenyl (NBS) stable isotope labeling: a new approach for the novel biomarker discovery for cancer

The recent progress in various proteomic technologies allows us to screen serum biomarker including carbohydrate antigens. However, only a limited number of proteins could be detected by current conventional methods such as shotgun proteomics, primarily because of the enormous concentration distribution of serum proteins and peptides. To circumvent this difficulty and isolate potential cancer-specific biomarkers for diagnosis and treatment, we established a new screening system consisting of the sequential steps of (1) immunodepletion of 6 high-abundance proteins, (2) targeted enrichment of glycoproteins by lectin column chromatography, and (3) the quantitative proteome analysis using 12C6- or 13C6-NBS (2-nitrobenzenesulfenyl) stable isotope labeling followed by MALDI-QIT-TOF mass spectrometric analysis. Through this systematic analysis for five serum samples derived from patients with lung adenocarcinoma, we identified as candidate biomarkers 34 serum glycoproteins that revealed significant difference in alpha1,6-fucosylation level between lung cancer and healthy control, clearly demonstrating that the carbohydrate-focused proteomics could allow for the detection of serum components with cancer-specific features. In addition, we developed a more simplified and practical technique, mass spectrometry-based glycan structure analysis and lectin blotting, in order to validate glycan structure of candidate biomarkers that could be applicable in clinical use. Our new glycoproteomic strategy will provide highly sensitive and quantitative profiling of specific glycan structures on multiple proteins, which should be useful for serum biomarker discovery.     

On the analysis of glycomics mass spectrometry data via the regularized area under the ROC curve

Background  

Novel molecular and statistical methods are in rising demand for disease diagnosis and prognosis with the help of recent advanced biotechnology. High-resolution mass spectrometry (MS) is one of those biotechnologies that are highly promising to improve health outcome. Previous literatures have identified some proteomics biomarkers that can distinguish healthy patients from cancer patients using MS data. In this paper, an MS study is demonstrated which uses glycomics to identify ovarian cancer. Glycomics is the study of glycans and glycoproteins. The glycans on the proteins may deviate between a cancer cell and a normal cell and may be visible in the blood. High-resolution MS has been applied to measure relative abundances of potential glycan biomarkers in human serum. Multiple potential glycan biomarkers are measured in MS spectra. With the objection of maximizing the empirical area under the ROC curve (AUC), an analysis method was considered which combines potential glycan biomarkers for the diagnosis of cancer.     

Sialic acids in T cell development and function

Virtually all cell surface proteins and many cell membrane lipids are glycosylated, creating a cell surface glycocalyx. The glycan chains attached to cell surface glycoproteins and glycolipids are complex structures with specific additions that determine functions of the glycans in cell–cell communication and cell sensing of the environment. One type of specific modification of cell surface glycans is decoration of glycan termini by sialic acids. On T cells, these terminal sialic acid residues are involved in almost every aspect of T cell fate and function, from cell maturation, differentiation, and migration to cell survival and cell death. The roles that sialylated glycans play in T cell development and function, including binding to specific sialic acid-binding lectins, are reviewed here.     

综述与专论: 肿瘤相关碳水化合物抗原的适配体研究

肿瘤细胞异常的糖基化模式是癌症的标志，在恶性转化和癌症进展中起着至关重要的作用。不同机制导致的肿瘤相关碳水化合物抗原（tumor-associated carbohydrate antigens，TACAs）不仅是临床肿瘤学诊断中公认的生物标志物，也为治疗干预提供了特定的靶点。适配体作为抗体或凝集素的有力替代品，近年来在碳水化合物的识别中展现了潜在的应用价值。本文聚焦于癌症中异常的糖基化改变，综述了目前TACAs识别适配体的开发进展。依据适配体筛选程序中的靶标来源，阐述了针对3种类型靶标，包括糖类分子、蛋白质聚糖表位，以及血清糖类抗原的筛选策略。从筛选方法、性能指标及相关应用性方面对适配体进行了总结，并讨论了当前研究中存在的问题和未来发展方向。     

Extraction of leukemia specific glycan motifs in humans by computational glycomics

There have been almost no standard methods for conducting computational analyses on glycan structures in comparison to DNA and proteins. In this paper, we present a novel method for extracting functional motifs from glycan structures using the KEGG/GLYCAN database. First, we developed a new similarity measure for comparing glycan structures taking into account the characteristic mechanisms of glycan biosynthesis, and we tested its ability to classify glycans of different blood components in the framework of support vector machines (SVMs). The results show that our method can successfully classify glycans from four types of human blood components: leukemic cells, erythrocyte, serum, and plasma. Next, we extracted characteristic functional motifs of glycans considered to be specific to each blood component. We predicted the substructure alpha-D-Neup5Ac-(2-->3)-beta-D-Galp-(1-->4)-D-GlcpNAc as a leukemia specific glycan motif. Based on the fact that the Agrocybe cylindracea galectin (ACG) specifically binds to the same substructure, we conducted an experiment using cell agglutination assay and confirmed that this fungal lectin specifically recognized human leukemic cells.     

Liquid Chromatography/Mass Spectrometry (LC/MS)-Based Glycoproteomics Technologies for Cancer Biomarker Discovery

Introduction

Biomarker discovery is a major objective of clinical proteomics; molecular biomarkers allow for detection of early-stage human diseases, especially cancer, and for monitoring their progression and/or regression after treatment. Biomarkers also help to elucidate the pathology of disease and its diagnosis, drug discovery, and toxicology. Glycans are ideal candidates for biomarkers because (1) glycoconjugates are localized on the cell surface and in the secretions such as plasma, (2) their structures are frequently and drastically changed during normal and aberrant cell differentiation, and (3) different cell types express different glycan signatures. Certain serodiagnostic glycoconjugate markers, such as carcinoembryonic antigen (CEA), are currently available; however, comprehensive glycome analysis has yet to be performed, mainly because of the difficulties of isolating and structurally analyzing complex glycans. Large-scale glycoprotein analysis, termed glycoproteomics, has the potential to effectively trace cellular glycoproteins and therefore to search for new serodiagnostic biomarkers.

Conclusions

In this review, we describe current mass spectrometry-based glycoproteomics technologies. Quantitative “shotgun” proteomics analyses of glycopeptides captured from complex biological mixtures such as plasma, coupled with advanced glycome technologies, enhance our knowledge of protein glycosylation and facilitate discovery of new biomarkers for human diseases.     

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.     

Identification of potential biomarkers for post-traumatic complications released after trauma–hemorrhage from murine Kupffer cells and its investigation in lung and liver

Context: Early diagnosis of complications after severe trauma by specific biomarkers remains difficult.

Objective: Identify potential new biomarkers for early diagnosis of post-traumatic complications.

Material and methods: Mice underwent pressure-controlled hemorrhage or sham procedure. Four hours later, genome-wide expression of isolated Kupffer cells was compared with controls using Affymetrix-Genechip-Expression-Analysis and real-time-PCR.

Results: Expression analysis and real-time-PCR revealed a significant increase of gene expression of Cxcl10, Il4ra, Csf2rb2, Lcn2, and Gbp5.

Conclusion: Cxcl10, Il4ra, Csf2rb2, Lcn2, and Gbp5 might represent new biomarkers for early diagnosis of post-traumatic complications, if they are linked to the development of post-traumatic complications.     

Impact of cultivation conditions on N‐glycosylation of influenza virus a hemagglutinin produced in MDCK cell culture

Manufacturers worldwide produce influenza vaccines in different host systems. So far, either fertilized chicken eggs or mammalian cell lines are used. In all these vaccines, hemagglutinin (HA) and neuraminidase are the major components. Both are highly abundant glycoproteins in the viral envelope, and particularly HA is able to induce a strong and protective immune response. The quality characteristics of glycoproteins, such as specific activity, antigenicity, immunogenicity, binding avidity, and receptor‐binding specificity can strongly depend on changes or differences in their glycosylation pattern (potential N‐glycosylation occupancy as well as glycan composition). In this study, capillary gel electrophoresis with laser‐induced fluorescence detection (CGE‐LIF) based glycoanalysis (N‐glycan fingerprinting) was used to determine the impact of cultivation conditions on the HA N‐glycosylation pattern of Madin–Darby canine kidney (MDCK) cell‐derived influenza virus A PR/8/34 (H1N1). We found that adaptation of adherent cells to serum‐free growth has only a minor impact on the HA N‐glycosylation pattern. Only relative abundances of N‐glycan structures are affected. In contrast, host cell adaptation to serum‐free suspension growth resulted in significant changes in the HA N‐glycosylation pattern regarding the presence of specific N‐glycans as well as their abundance. Further controls such as different suppliers for influenza virus A PR/8/34 (H1N1) seed strains, different cultivation scales and vessels in standard or high cell density mode, different virus production media varying in either composition or trypsin activity, different temperatures during virus replication and finally, the impact of β‐propiolactone inactivation resulted—at best—only in minor changes in the relative N‐glycan structure abundances of the HA N‐glycosylation pattern. Surprisingly, these results demonstrate a rather stable HA N‐glycosylation pattern despite various (significant) changes in upstream processing. Only the adaptation of the production host cell line to serum‐free suspension growth significantly influenced HA N‐glycosylation regarding both, the type of attached glycan structures as well as their abundances. Biotechnol. Bioeng. 2013; 110: 1691–1703. © 2013 Wiley Periodicals, Inc.     

Deciphering the glycocode: the complexity and analytical challenge of glycomics

Carbohydrates coat most types of cell in nature and are intimately involved in various biological events, including cell differentiation, homing to specific tissues, cell adhesion, cell recognition, microbial pathogenesis and immunological recognition. Carbohydrate structures are complex to analyze owing to their branched nature, the diversity of secondary modifications of monomers, their indirect relationship to the genome and the range of molecular contexts in which the modifications are found. Thus, whereas the fields of genomics and proteomics have become accessible to most scientists, technologies to assess glycan structures rapidly (i.e. glycomics) are still in the developmental stages. This review focuses on recent developments in glycomic technologies, including new high-throughput techniques for glycan purification and annotation that are advancing mass-spectrometry-based glycomics, and the latest work on microarray methodologies to decipher the glycome.     

Potentials of single‐cell biology in identification and validation of disease biomarkers

Single‐cell biology is considered a new approach to identify and validate disease‐specific biomarkers. However, the concern raised by clinicians is how to apply single‐cell measurements for clinical practice, translate the message of single‐cell systems biology into clinical phenotype or explain alterations of single‐cell gene sequencing and function in patient response to therapies. This study is to address the importance and necessity of single‐cell gene sequencing in the identification and development of disease‐specific biomarkers, the definition and significance of single‐cell biology and single‐cell systems biology in the understanding of single‐cell full picture, the development and establishment of whole‐cell models in the validation of targeted biological function and the figure and meaning of single‐molecule imaging in single cell to trace intra‐single‐cell molecule expression, signal, interaction and location. We headline the important role of single‐cell biology in the discovery and development of disease‐specific biomarkers with a special emphasis on understanding single‐cell biological functions, e.g. mechanical phenotypes, single‐cell biology, heterogeneity and organization of genome function. We have reason to believe that such multi‐dimensional, multi‐layer, multi‐crossing and stereoscopic single‐cell biology definitely benefits the discovery and development of disease‐specific biomarkers.     

Development of Serum Glycoproteomic Profiling Technique; Simultaneous Identification of Glycosylation Sites and Site-Specific Quantification of Glycan Structure Changes

Characterization and interpretation of disease-associated alterations of protein glycosylation are the central aims of the emerging glycoproteomics projects, which are expected to lead to more sensitive and specific diagnosis and improve therapeutic outcomes for various diseases. Here we report a new approach to identify carbohydrate-targeting serum biomarkers, termed isotopic glycosidase elution and labeling on lectin-column chromatography (IGEL). This technology is based on glycan structure-specific enrichment of glycopeptides by lectin-column chromatography and site-directed tagging of N-glycosylation sites by ¹⁸O during the elution with N-glycosidase. The combination of IGEL with 8-plex isobaric tag for relative and absolute quantitation (iTRAQ) stable isotope labeling enabled us not only to identify N-glycosylation sites effectively but also to compare glycan structures on each glycosylation site quantitatively in a single LC/MS/MS analysis. We applied this method to eight sera from lung cancer patients and controls, and finally identified 107 glycopeptides in their sera, including A2GL_Asn151, A2GL_Asn290, CD14_Asn132, CO8A_Asn417, C163A_Asn64, TIMP1_Asn30, and TSP1_Asn1049 which showed the significant change of the affinity to Concanavalin A (ConA) lectin between the lung cancer samples and the controls (p < 0.05 and more than twofold change). These screening results were further confirmed by the conventional lectin-column chromatography and immunoblot analysis using additional serum samples. Our novel methodology, which should be valuable for diverse biomarker discoveries, can provide high-throughput and quantitative profiling of glycan structure alterations.Glycan structure variations often show highly organ-specific manners (1, 2), as well as those manners that correlate with diverse disease states, (3, 4) e.g., cancer and inflammation. Thus, the carbohydrates are currently attracting a great deal of attention as specific targets of cancer biomarkers and therapy (5). In fact, certain changes of glycan structures are already in clinical use as serum biomarkers, such as AFP-L3 (6), and glycosylation at the specific site of therapeutic antibody proved to be essential for its therapeutic effect (7). Advances in proteomic technologies and analysis have stimulated a great interest in application of MS to identify glycosylation sites (8, 9) or analyze glycan structures (10, 11) from various biological specimens, but the comprehensive techniques which allow quantitative profiling of glycan structures on each glycosylation site have not been developed.The two major issues facing recent glycoproteomic studies are the difficulties in glycopeptide-specific enrichment tools involving lectin-column chromatography and the detection of glycopeptides in mass spectrometers. In the conventional lectin-column chromatography experiments, glycoprotein enrichment from complicated protein mixtures, such as human sera, resulted in a heavy contamination of hapten sugar, salts, and nonspecific proteins caused by protein-protein interactions of serum proteins (12). Even when the digested peptide mixture was subjected to the lectin-column chromatography, salt contamination and the eluting sugar-dependent biases of elution efficiency were inevitable. Moreover, the straightforward analysis of the eluted glycopeptides by MS was hardly possible without further deglycosylation and desalting steps.In this study we report our new approach for the identification of carbohydrate-targeting biomarkers, termed isotopic glycosidase elution and labeling on lectin-column chromatography (IGEL),¹ which is based on glycan structure-specific enrichment of glycopeptides by lectin- column chromatography and site-directed labeling of N-glycosylation sites by water-¹⁸O during the elution with N-glycosidase. We combined this method with 8-plex isotobaric tag for relative and absolute quantitation (iTRAQ) labeling for relative quantification of glycopeptides and applied them to search for carbohydrate-targeting serum biomarkers in lung cancer patient sera.     

Evaluation of the serum N-linked glycome for the diagnosis of cancer and chronic inflammation

The identification of serum biomarkers has lead to improvements in the detection and diagnosis of cancer, and combinations of these biomarkers have increased further their sensitivity and specificity. Glycosylation is the most common PTM of secreted proteins and the identification of novel serum glyco-biomarkers has become a topic of increasing interest because the glycan processing pathways are frequently disturbed in cancer cells. A future goal is to combine current biomarkers with glyco-biomarkers to yield further improvements. Well characterised N-glycosylation changes in the serum glycome of cancer patients include changes in the levels of tri- and tetra-antennary glycan structures, sialyl Lewis X epitopes and agalactosylated bi-antennary glycans. Several of these glycosylated markers have been linked to chronic inflammatory diseases, promoting questions about the links between inflammation and cancer. In this review, the glycoproteins which display these glycan epitopes, the glycosyl transferases which can generate them, their potential functions and their use as biomarkers are evaluated.     

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.