Tetra-antennary N-glycans Research Articles

High-abundance serum glycoproteins as valuable resources for glycopeptide standards

High-abundance serum proteins, mostly modified by N-glycans, are usually depleted from human sera to achieve in-depth analyses of serum proteome and sub-proteomes. In this study, we show that these high-abundance glycoproteins (HAGPs) can be used as valuable standard glycopeptide resources, as long as the structural features of their glycans have been well defined at the glycosite-specific level. By directly analyzing intact glycopeptides enriched from serum, we identified 1322 unique glycopeptides at 48 N-glycosites from the top 12 HAGPs (19 subclasses). These HAGPs could be further classified into four major groups based on the structural features of their attached N-glycans. Immunoglobins including IGHG1/2/3/4, IGHA1/2 and IGHM were mostly modified by core fucosylated and bisected N-glycans with rarely sialic acids. Alpha-1-acid glycoproteins (ORM1/2) and haptoglobins (HP) were mainly modified by tri-and tetra-antennary (40 %) N-glycans with antenna-fucoses and sialic acids. Complement components C3 and C4A/B were highly modified by oligo-mannose glycans. The other HAGPs including SERPINA1, A2M, TF, FGB/G and APOB mainly contain bi-antennary complex glycans with the common core structure and (sialyl-) LacNAc branch structures. These HAGPs are easily detected by LC-MS analysis and therefore could be used as standard glycopeptides for glycoproteomic methodology studies as well as possible clinical utilities.

Galactosylation of glycoconjugates using Pacific oyster β-1,3-galactosyltransferases

The Pacific oyster (Magallana gigas) exhibits an extensive diversity of N- and O-linked glycoconjugates, offering significant potential for biotechnological applications. Through genomic data mining, we have identified and characterized a suite of β-1,3-galactosyltransferase enzymes, pivotal for the synthesis of glycan structures. Out of ten cloned gene candidates, six enzymes were successfully expressed recombinantly in Escherichia coli. Four of these enzymes exhibited measurable catalytic activity in the transfer of galactose to various acceptor substrates. Notably, MgB3GalT1 demonstrated the highest efficiency, achieving a 91.2 % conversion rate. This enzyme was proficient in glycosylating diverse glycan structures, including Core 2 O-glycans and several di-, tri-, and tetra-antennary complex N-glycan standards. Mass spectrometric analysis confirmed the successful modification of N-glycans. These findings open new approaches for utilizing oyster-derived enzymes in glycan-based therapeutics and molecular glycoengineering, highlighting their utility in synthetic applications and biotechnological advancements.

Utilizing multimodal mass spectrometry imaging for profiling immune cell composition and N-glycosylation across colorectal carcinoma disease progression.

Colorectal cancer (CRC) stands as a leading cause of death worldwide, often arising from specific genetic mutations, progressing from pre-cancerous adenomas to adenocarcinomas. Early detection through regular screening can result in a 90% 5-year survival rate for patients. However, unfortunately, only a fraction of CRC cases are identified at pre-invasive stages, allowing progression to occur silently over 10-15years. The intricate interplay between the immune system and tumor cells within the tumor microenvironment plays a pivotal role in the progression of CRC. Immune cell clusters can either inhibit or facilitate tumor initiation, growth, and metastasis. To gain a better understanding of this relationship, we conducted N-glycomic profiling using matrix-assisted laser desorption-ionization mass spectrometry imaging (MALDI-MSI). We detected nearly 100 N-glycan species across all samples, revealing a shift in N-glycome profiles from normal to cancerous tissues, marked by a decrease in high mannose N-glycans. Further analysis of precancerous to invasive carcinomas showed an increase in pauci-mannose biantennary, and tetraantennary N-glycans with disease progression. Moreover, a distinct stratification in the N-glycome profile was observed between non-mucinous and mucinous CRC tissues, driven by pauci-mannose, high mannose, and bisecting N-glycans. Notably, we identified immune clusters of CD20+ B cells and CD3/CD44+ T cells distinctive and predictive with signature profiles of bisecting and branched N-glycans. These spatial N-glycan profiles offer potential biomarkers and therapeutic targets throughout the progression of CRC.

N-glycan profiling of tissue samples to aid breast cancer subtyping

Breast cancer is a highly heterogeneous disease. Its intrinsic subtype classification for diagnosis and choice of therapy traditionally relies on the presence of characteristic receptors. Unfortunately, this classification is often not sufficient for precise prediction of disease prognosis and treatment efficacy. The N-glycan profiles of 145 tumors and 10 healthy breast tissues were determined using Matrix-Assisted Laser Desorption-Ionization Time-of-Flight Mass Spectrometry. The tumor samples were classified into Mucinous, Lobular, No-Special-Type, Human Epidermal Growth Factor 2 + , and Triple-Negative Breast Cancer subtypes. Statistical analysis was conducted using the reproducibility-optimized test statistic software package in R, and the Wilcoxon rank sum test with continuity correction. In total, 92 N-glycans were detected and quantified, with 59 consistently observed in over half of the samples. Significant variations in N-glycan signals were found among subtypes. Mucinous tumor samples exhibited the most distinct changes, with 28 significantly altered N-glycan signals. Increased levels of tri- and tetra-antennary N-glycans were notably present in this subtype. Triple-Negative Breast Cancer showed more N-glycans with additional mannose units, a factor associated with cancer progression. Individual N-glycans differentiated Human Epidermal Growth Factor 2 + , No-Special-Type, and Lobular cancers, whereas lower fucosylation and branching levels were found in N-glycans significantly increased in Luminal subtypes (Lobular and No-Special-Type tumors). Clinically normal breast tissues featured a higher abundance of signals corresponding to N-glycans with bisecting moiety. This research confirms that histologically distinct breast cancer subtypes have a quantitatively unique set of N-glycans linked to clinical parameters like tumor size, proliferative rate, lymphovascular invasion, and metastases to lymph nodes. The presented results provide novel information that N-glycan profiling could accurately classify human breast cancer samples, offer stratification of patients, and ongoing disease monitoring.

Recognition of Highly Branched N-Glycans of the Porcine Whipworm by the Immune System

Glycans are key to host–pathogen interactions, whereby recognition by the host and immunomodulation by the pathogen can be mediated by carbohydrate binding proteins, such as lectins of the innate immune system, and their glycoconjugate ligands. Previous studies have shown that excretory-secretory products of the porcine nematode parasite Trichuris suis exert immunomodulatory effects in a glycan-dependent manner. To better understand the mechanisms of these interactions, we prepared N-glycans from T. suis and both analyzed their structures and used them to generate a natural glycan microarray. With this array, we explored the interactions of glycans with C-type lectins, C-reactive protein, and sera from T. suis–infected pigs. Glycans containing LacdiNAc and phosphorylcholine-modified glycans were associated with the highest binding by most of these proteins. In-depth analysis revealed not only fucosylated LacdiNAc motifs with and without phosphorylcholine moieties but phosphorylcholine-modified mannose and N-acetylhexosamine–substituted fucose residues, in the context of maximally tetraantennary N-glycan scaffolds. Furthermore, O-glycans also contained fucosylated motifs. In summary, the glycans of T. suis are recognized by both the innate and adaptive immune systems and also exhibit species-specific features distinguishing its glycome from those of other nematodes.

Complex-Type N-Glycans Are Associated with Cartilage Degeneration within Different Loading Sites of the Tibial Plateau for Knee Osteoarthritis Patients.

Abnormal N-glycosylation has been shown to play an important role in the pathogenesis of multiple diseases. However, little is known about the relationship between N-glycosylation and knee osteoarthritis (KOA) progression at the tissue level. Thus, the aim of this study was to quantify the cartilage histomorphometric changes in formalin-fixed paraffin-embedded (FFPE) tissue collected from the lateral and medial compartments of the tibial plateau KOA patients (n = 8). Subsequently, N-glycans were analyzed by matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) followed by in situ MS/MS fragmentation. Overall, the Osteoarthritis Research Society International (OARSI) histological grade and cartilage surface fibrillation index were significantly higher, and chondrocyte size in the superficial zone was much larger, for the medial high-loaded cartilage compared to the lateral less-loaded cartilage. Among 92 putative N-glycans observed by MALDI-MSI, 3 complex-type N-glycans, (Hex)4(HexNAc)3, (Hex)4(HexNAc)4, and (Hex)5(HexNAc)4, and 1 oligomannose-type N-glycan, (Hex)9(HexNAc)2, were significantly higher in intensity in the medial cartilage compared to the lateral cartilage, whereas 2 tetra-antennary fucosylated-type N-glycans, (Hex)3(HexNAc)6(Fuc)2 and (Hex)3(HexNAc)6(Fuc)3, were significantly higher in intensity in the lateral cartilage than the medial cartilage. Our findings indicate that complex-type N-glycans are associated with higher severity of cartilage degeneration and may influence the cellular processes of KOA.

Serum N-Glycosylation RPLC-FD-MS Assay to Assess Colorectal Cancer Surgical Interventions.

A newly developed analytical strategy was applied to profile the total serum N-glycome of 64 colorectal cancer (CRC) patients before and after surgical intervention. In this cohort, it was previously found that serum N-glycome alterations in CRC were associated with patient survival. Here, fluorescent labeling of serum N-glycans was applied using procainamide and followed by sialic acid derivatization specific for α2,6- and α2,3-linkage types via ethyl esterification and amidation, respectively. This strategy allowed efficient separation of specific positional isomers on reversed-phase liquid chromatography-fluorescence detection-mass spectrometry (RPLC-FD-MS) and complemented the previous glycomics data based on matrix-assisted laser desorption/ionization (MALDI)-MS that did not include such separations. The results from comparing pre-operative CRC to post-operative samples were in agreement with studies that identified a decrease in di-antennary structures with core fucosylation and an increase in sialylated tri- and tetra-antennary N-glycans in CRC patient sera. Pre-operative abundances of N-glycans showed good performance for the classification of adenocarcinoma and led to the revisit of the previous MALDI-MS dataset with regard to histological and clinical data. This strategy has the potential to monitor patient profiles before, during, and after clinical events such as treatment, therapy, or surgery and should also be further explored.

N-glycosylation reinforces interaction of immune checkpoint TIM-3 with a small molecule ligand

N-glycosylation of eukaryotic proteins plays roles in protein folding, trafficking, and signal transduction. The biological influence of the process is well understood, whereas the pharmacological impact of protein N-glycosylation is not well under discerned. The role of N-glycosylation on drug binding to protein has been rarely studied. We have modeled the influence of a bi-antennary N-glycan introduced at position N78 on the immune checkpoint TIM-3 (T cell immunoglobulin domain and mucin domain-containing molecule 3) on the interaction with a selective drug antagonist. The bulky N-glycan introduced at the consensus sequence Asn-Val-Thr has no influence on drug binding when the glycan adopts an extended conformation. But in a folded conformation, the glycan can interact directly with the triazoloquinazolinone derivative so as to further stabilize the drug-TIM-3 complex. The non-fucosylated glycan at position N78 markedly consolidates the drug interaction, via an additional H-bond interaction with the α3-mannose residue. It provides a gain of empirical potential energy of interaction (ΔE) of about 30 %. The presence of a more rigid fucosylated N-glycan is a little less favorable, with a gain of ΔE of about 20 %. The folded N-glycan appears to protect the ligand bound to the protein cavity, with the tricyclic core of the heterocyclic molecule sandwiched between two indole rings of tryptophan residues. Similar results were obtained when using a biantennary disialyl N-glycan with a bisecting GlcNAc residue and a tetra-antennary N-glycan. The molecular models illustrate the drug-stabilizing capacity of a bulky N-glycan positioned at a validated glycosylation site (N78 corresponding to N100 for the full-length protein). The modeling approach is useful to delineate further the role of the N-glycan of the immune checkpoint TIM-3 in interaction with small molecule ligands, and to guide the design of more potent compounds. The approach is transposable to other proteins to better comprehend the influence of N-glycans on drug-receptor interactions.

Site‐specific N‐glycan profiles of α5β1 integrin from rat liver

Like most other cell surface proteins, α5 β1 integrin is glycosylated, which is required for its various activities in ways that mostly remain to be determined. Here, we have established the first comprehensive site-specific glycan map of α5 β1 integrin that was purified from a natural source, that is, rat liver. This analysis revealed striking site selective variations in glycan composition. Complex bi, tri, or tetraantennary N-glycans were predominant at various proportions at most potential N-glycosylation sites. A few of these sites were nonglycosylated or contained high mannose or hybrid glycans, indicating that early N-glycan processing was hindered. Almost all complex N-glycans had fully galactosylated and sialylated antennae. Moderate levels of core fucosylation and high levels of O-acetylation of NeuAc residues were observed at certain sites. An O-linked HexNAc was found in an EGF-like domain of β1 integrin. The extensive glycan information that results from our study was projected onto a map of α5 β1 integrin that was obtained by homology modeling. We have used this model for the discussion of how glycosylation might be used in the functional cycle of α5 β1 integrin. A striking example concerns the involvement of glycan-binding galectins in the regulation of the molecular homeostasis of glycoproteins at the cell surface through the formation of lattices or endocytic pits according to the glycolipid-lectin (GL-Lect) hypothesis. We expect that the glycoproteomics data of the current study will serve as a resource for the exploration of structural mechanisms by which glycans control α5 β1 integrin activity and endocytic trafficking. Glycosylation of α5 β1 integrin has been implicated in multiple aspects of integrin function and structure. Yet, detailed knowledge of its glycosylation, notably the specific sites of glycosylation, is lacking. Furthermore, the α5 β1 integrin preparation that was analyzed here is from a natural source, which is of importance as there is not a lot of literature in the field about the glycosylation of "native" glycoproteins.

Conformational preferences of triantennary and tetraantennary hybrid N-glycans in aqueous solution: Insights from 20 μs long atomistic molecular dynamic simulations

In the current study, we have investigated the conformational dynamics of a triantennary (N-glycan1) and tetraantennary (N-glycan2) hybrid N-glycans found on the surface of the HIV glycoprotein using 20 μs long all-atom molecular dynamics (MD) simulations. The main objective of the present study is to elucidate the influence of adding a complex branch on the overall glycan structural dynamics. Our investigation suggests that the average RMSD value increases when a complex branch is added to N-glycan1. However, the RMSD distribution is relatively wider in the case of N-glycan1 compared to N-glycan2, which indicates that multiple complex branches restrict the conformational variability of glycans. A similar observation is obtained from the principal component analysis of both glycans. All the puckering states (4C1 to 1C4) of each monosaccharide except mannose are sampled in our simulations, although the 4C1 chair form is energetically more favorable than 1C4. In N-glycan1, the 1–6 linkage in the mannose branch [Man(9)-α(1-6)-Man(5)] stays in the gauche-gauche cluster, whereas it moves towards trans-gauche in N-glycan2. For both glycans, mannose branches are more flexible than the complex branches, and adding a complex branch does not influence the dynamics of the mannose branches. We have noticed that the end-to-end distance of the complex branch shortens by ∼ 10 Å in the presence of another complex branch. This suggests that in the presence of an additional complex branch, the other complex branch adopts a close folded structure. All these conformational changes involve the selective formation of inter-residue and water-mediated hydrogen-bond networks.

O ‐GlcNAcylation regulates β1,4‐GlcNAc‐branched N ‐glycan biosynthesis via the OGT/SLC35A3/GnT‐IV axis

N-Linked glycosylation and O-linked N-acetylglucosamine (O-GlcNAc) are important protein post-translational modifications that are orchestrated by a diverse set of gene products. Thus far, the relationship between these two types of glycosylation has remained elusive, and it is unclear whether one influences the other via UDP-GlcNAc, which is a common donor substrate. Theoretically, a decrease in O-GlcNAcylation may increase the products of GlcNAc-branched N-glycans. In this study, via examination by lectin blotting, HPLC, and mass spectrometry analysis, however, we found that the amounts of GlcNAc-branched tri-antennary N-glycans catalyzed by N-acetylglucosaminyltransferase IV (GnT-IV) and tetra-antennary N-glycans were significantly decreased in O-GlcNAc transferase knockdown cells (OGT-KD) compared with those in wild type cells. We examined this specific alteration by focusing on SLC35A3, which is the main UDP-GlcNAc transporter in mammals that is believed to modulate GnT-IV activation. It is interesting that a deficiency of SLC35A3specifically leads to a decrease in the amounts of GlcNAc-branched tri- and tetra-antennary N-glycans. Furthermore, co-immunoprecipitation experiments have shown that SLC35A3 interacts with GnT-IV, but not with N-acetylglucosaminyltransferase V. Western blot and chemoenzymatic labeling assay have confirmed that OGT modifies SLC35A3 and that O-GlcNAcylation contributes to its stability. Furthermore, we found that SLC35A3-KO enhances cell spreading and suppresses both cell migration and cell proliferation, which is similar to the phenomena observed in the OGT-KD cells. Taken together, these data are the first to demonstrate that O-GlcNAcylation specifically governs the biosynthesis of tri- and tetra-antennary N-glycans via the OGT-SLC35A3-GnT-IV axis.

Chemical Synthesis of Sialyl N-Glycans and Analysis of Their Recognition by Neuraminidase.

The chemical synthesis of a fully sialylated tetraantennary N-glycan has been achieved for the first time by using the diacetyl strategy, in which NHAc is protected as NAc2 to improve reactivity by preventing intermolecular hydrogen bonds. Another key was the glycosylation to the branched mannose in an ether solvent, which promoted the desired glycosylation by stabilizing the oxocarbenium ion intermediate. Furthermore, high α-selectivity of these glycosylation reactions was realized by utilizing remote participation. Two asymmetrically deuterium labeled sialyl N-glycans were also synthesized by the same strategy. The synthesized N-glycans were used to probe the molecular basis of H1N1 neuraminidase recognition. The asymmetrically deuterated N-glycans revealed a difference in the recognition of sialic acid on each branch. Meanwhile, the tetraantennary N-glycan was used to evaluate the effects of multivalency and steric hinderance by forming branching structures.

FUT8-Directed Core Fucosylation of N-glycans Is Regulated by the Glycan Structure and Protein Environment

FUT8 is an essential α-1,6-fucosyltransferase that fucosylates the innermost GlcNAc of N-glycans, a process called core fucosylation. In vitro, FUT8 exhibits substrate preference for the biantennary complex N-glycan oligosaccharide (G0), but the role of the underlying protein/peptide to which N-glycans are attached remains unclear. Here, we explored the FUT8 enzyme with a series of N-glycan oligosaccharides, N-glycopeptides, and an Asn-linked oligosaccharide. We found that the underlying peptide plays a role in fucosylation of paucimannose (low mannose) and high-mannose N-glycans but not for complex-type N-glycans. Using saturation transfer difference (STD) NMR spectroscopy, we demonstrate that FUT8 recognizes all sugar units of the G0 N-glycan and most of the amino acid residues (Asn-X-Thr) that serve as a recognition sequon for the oligosaccharyltransferase (OST). The largest STD signals were observed in the presence of GDP, suggesting that prior FUT8 binding to GDP-β-l-fucose (GDP-Fuc) is required for an optimal recognition of N-glycans. We applied genetic engineering of glycosylation capacities in CHO cells to evaluate FUT8 core fucosylation of high-mannose and complex-type N-glycans in cells with a panel of well-characterized therapeutic N-glycoproteins. This confirmed that core fucosylation mainly occurs on complex-type N-glycans, although clearly only at selected glycosites. Eliminating the capacity for complex-type glycosylation in cells (KO mgat1) revealed that glycosites with complex-type N-glycans when converted to high mannose lost the core Fuc. Interestingly, however, for erythropoietin that is uncommon among the tested glycoproteins in efficiently acquiring tetra-antennary N-glycans, two out of three N-glycosites obtained Fuc on the high-mannose N-glycans. An examination of the N-glycosylation sites of several protein crystal structures indicates that core fucosylation is mostly affected by the accessibility and nature of the N-glycan and not by the nature of the underlying peptide sequence. These data have further elucidated the different FUT8 acceptor substrate specificities both in vitro and in vivo in cells, revealing different mechanisms for promoting core fucosylation.

N-glycan structures of Wisteria floribunda agglutinin-positive Mac2 binding protein in the serum of patients with liver fibrosis

The extent of liver fibrosis predicts prognosis and is important for determining treatment strategies for chronic hepatitis. During the fibrosis progression, serum levels of Mac2 binding protein (M2BP) increase and the N-glycan structure changes to enable binding to Wisteria floribunda agglutinin (WFA) lectin. As a novel diagnostic marker, glycosylation isomer of M2BP (M2BPGi) has been developed. However, its glycan structures recognized by WFA are unclear. In this study, we analyzed site-specific N-glycan structures of serum M2BP using Glyco-RIDGE (Glycan heterogeneity-based Relational IDentification of Glycopeptide signals on Elution profile) method. We evaluated five sample types: (1) M2BP immunoprecipitated from normal healthy sera (NHS-IP(+)), (2) M2BP immunoprecipitated from sera of patients with liver cirrhosis (stage 4; F4-IP(+)), (3) M2BP captured with WFA from serum of patients with liver cirrhosis (stage 4; F4-WFA(+)), (4) recombinant M2BP produced by HEK293 cells (rM2BP) and (5) WFA-captured rM2BP (rM2BP-WFA(+)). In NHS-IP(+) M2BP, bi-antennary N-glycan was the main structure, and LacNAc extended to its branches. In F4-IP(+) M2BP, many branched structures, including tri-antennary and tetra-antennary N-glycans, were found. F4-WFA(+) showed a remarkable increase in branched structures relative to the quantity before enrichment. In recombinant M2BP, both no sialylated-LacdiNAc and -branched LacNAc structures were emerged. The LacdiNAc structure was not found in serum M2BP. Glycosidase-assisted HISCL assays suggest that reactivity with WFA of both serum and recombinant M2BP depends on unsialylated and branched LacNAc and in part of recombinant depends on LacdiNAc. On M2BPGi, the highly branched LacNAc, probably dense cluster of LacNAc, would be recognized by WFA.

Preparation and biological activities of anti-HER2 monoclonal antibodies with multibranched complex-type N-glycans.

Immunoglobulin G (IgG) has a conserved N-glycosylation site at Asn297 in the fragment crystallizable (Fc) region. Previous studies have shown that N-glycosylation of this site is a critical mediator of the antibody's effector functions, such as antibody-dependent cellular cytotoxicity. While the N-glycan structures attached to the IgG-Fc region are generally heterogenous, IgGs engineered to be homogenously glycosylated with functional N-glycans may improve the efficacy of antibodies. The major glycoforms of the N-glycans on the IgG-Fc region are bi-antennary complex-type N-glycans, while multibranched complex-type N-glycans are not typically found. However, IgGs with tri-antennary complex-type N-glycans have been generated using the N-glycan remodeling technique, suggesting that more branched N-glycans might be artificially attached. At present, little is known about the properties of these IgGs. In this study, IgGs with multibranched N-glycans on the Fc region were prepared by using a combination of the glycosynthase/oxazoline substrate-based N-glycan remodeling technique and successive reactions with glycosyltransferases. Among the IgGs produced by these methods, the largest N-glycan attached was a bisecting N-acetylglucosamine containing a sialylated penta-antennary structure. Concerning the Fc-mediated effector functions, the majority of IgGs with tri- and tetra-antennary N-glycans on their Fc region showed properties similar to IgGs with ordinary bi-antennary N-glycans.

In Situ Analysis of N-Linked Glycans as Potential Biomarkers of Clinical Course in Human Prostate Cancer.

Prostate cancer is the most common cancer in men worldwide. Despite its prevalence, there is a critical knowledge gap in understanding factors driving disparities in survival among different cohorts of patients with prostate cancer. Identifying molecular features separating disparate populations is an important first step in prostate cancer research that could lead to fundamental hypotheses in prostate biology, predictive biomarker discovery, and personalized therapy. N-linked glycosylation is a cotranslational event during protein folding that modulates a myriad of cellular processes. Recently, aberrant N-linked glycosylation has been reported in prostate cancers. However, the full clinical implications of dysregulated glycosylation in prostate cancer has yet to be explored. Herein, we performed direct on-tissue analysis of N-linked glycans using matrix-assisted laser desorption ionization-mass spectrometry imaging (MALDI-MSI) from tissue microarrays of over 100 patient tumors with over 10 years of follow-up metadata. We successfully identified a panel of N-glycans that are unique between benign and prostate tumor tissue. Specifically, high-mannose as well as tri-and tetra-antennary N-glycans were more abundant in tumor tissue and increase proportionally with tumor grade. Further, we expanded our analyses to examine the N-glycan profiles of Black and Appalachian patients and have identified unique glycan signatures that correlate with recurrence in each population. Our study highlights the potential applications of MALDI-MSI for digital pathology and biomarker discovery for prostate cancer. IMPLICATIONS: MALDI-MSI identifies N-glycan perturbations in prostate tumors compared with benign tissue. This method can be utilized to predict prostate cancer recurrence and study prostate cancer disparities.

Abstract PO-094: Mass spectrometry imaging of N-glycans identifies racial discrepancies in human prostate tumors

Abstract Prostate cancer is the most commonly diagnosed cancer in men worldwide. A critical knowledge gap in prostate cancer biology is the molecular events underlining higher incidence and mortality rate in Black men. Identifying molecular features that separate racial disparities is a critical step in prostate cancer research that could lead to predictive biomarkers and personalized therapy. N-linked glycosylation is a required co-translational event during protein folding that modulates many biological processes, such as cell adhesion, immune modulation, cell-matrix interactions, and cell proliferation. Recently, aberrant N-linked glycosylation has been reported in prostate cancers. However, the full clinical implications of dysregulated glycosylation in prostate cancer has yet to be explored. Matrix-assisted laser desorption/ionization-mass spectrometry imaging (MALDI-MSI) is a new and innovative technique that combines mass spectrometry with imaging, enabling the detection of glycans with spatial distribution. Herein, we performed MALDI-MSI analysis to characterize the N-glycan profile from tissue microarrays of over 100 patient tumors banked at the University of Kentucky with over 10 years of follow-up data. Additionally, we performed MALDI-MSI analysis on large sections of prostate cancer to define the regional distribution of N-glycans within the tumor microenvironment. We successfully identified 46 unique glycans from readily available formalin-fixed paraffin-embedded prostate tissue, and found significant N- glycan dysregulation between benign and prostate tumor tissue across all patient groups. High mannose as well as tri- and tetra-antennary N-glycans were predominantly found in tumor tissue and correlate with increased tumor grade. Surprisingly, several species of N-glycans were profoundly different between early grade prostate tumors resected from White and Black patients. Further, these glycans predict opposing overall survival between White and Black patients with prostate cancer. These data suggest differential N-linked glycosylation underline the racial disparity of prostate cancer prognosis. Our study highlights the potential clinical applications of MALDI-MSI for digital pathology and biomarker applications, and reveals molecular features that contribute to the racial disparity in diagnosis and survival of prostate cancer patients. Citation Format: Lindsey R. Conroy, Lyndsay E.A. Young, Grant L. Austin, Alexanda E. Stanback, Derek B Allison, Matthew S. Gentry, Richard R. Drake, Ramon C. Sun. Mass spectrometry imaging of N-glycans identifies racial discrepancies in human prostate tumors [abstract]. In: Proceedings of the AACR Virtual Conference: Thirteenth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2020 Oct 2-4. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2020;29(12 Suppl):Abstract nr PO-094.

In vivo modification of the goat mammary gland glycosylation pathway

Complex recombinant glycoproteins produced as potential biopharmaceuticals in goat’s milk have an aberrant pattern of N-glycosylation due to the lack of multi-antennary structures. Overexpression of glycosyltransferases may increase oligosaccharide branching of the desired glycoproteins. Here, human erythropoietin fused to human IgG Fc (EPO-Fc) was co-expressed with N-acetyl-glucosaminyltransferase-IVa (GnT-IVa) by adenoviral transduction in goat mammary gland to evaluate the in vivo modification of N-glycosylation pattern in this tissue. Adenoviral vectors, containing the EPO-Fc and GnT-IVa sequences were assembled for in vitro and in vivo expression in mammalian cell culture or in goat mammary gland. Protein detection was assessed by gel electrophoresis and western blot, and N-glycans were identified by HPLC and mass spectrometry. GnT-IVa overexpression and its colocalization with EPO-Fc in the Golgi apparatus of SiHa cells were demonstrated. N-glycan analysis of in vitro and in vivo expression of EPO-Fc modified by GnT-IVa (EPO-Fc/GnT-IVa) showed an increase in high molecular weight structures, which corresponded to tri- and tetra-antennary N-glycans in SiHa cells and mostly tri-antennary N-glycans in goat’s milk from transformed mammary tissue. The results confirmed that successful modification of the goat mammary gland secretion pathway could be achieved by co-expressing glycoenzymes together with the glycoprotein of interest. This is the first report of modification of the N-glycosylation pattern in the goat mammary gland in vivo, and constitutes a step forward for improving the use of the mammary gland as a bioreactor for the production of complex recombinant proteins.

Abstract PO-085: Mass spectrometry imaging of N-glycans identifies racial discrepancies in human prostate tumors

Abstract Prostate cancer is the most commonly diagnosed cancer in men worldwide. A critical knowledge gap in prostate cancer biology is the molecular events underlining higher incidence and mortality rate in Black men. Identifying molecular features that separate racial disparities is a critical step in prostate cancer research that could lead to predictive biomarkers and personalized therapy. N-linked glycosylation is a required co-translational event during protein folding that modulates many biological processes, such as cell adhesion, immune modulation, cell-matrix interactions, and cell proliferation. Recently, aberrant N-linked glycosylation has been reported in prostate cancers. However, the full clinical implications of dysregulated glycosylation in prostate cancer has yet to be explored. Matrix-assisted laser desorption/ionization-mass spectrometry imaging (MALDI-MSI) is a new and innovative technique that combines mass spectrometry with imaging, enabling the detection of glycans with spatial distribution. Herein, we performed MALDI-MSI analysis to characterize the N-glycan profile from tissue microarrays of over 100 patient tumors banked at the University of Kentucky with over 10 years of follow-up data. Additionally, we performed MALDI-MSI analysis on large sections of prostate cancer to define the regional distribution of N-glycans within the tumor microenvironment. We successfully identified 46 unique glycans from readily available formalin-fixed paraffin-embedded prostate tissue, and found significant N-glycan dysregulation between benign and prostate tumor tissue across all patient groups. High mannose as well as tri- and tetra-antennary N-glycans were predominantly found in tumor tissue and correlate with increased tumor grade. Surprisingly, several species of N-glycans were profoundly different between early grade prostate tumors resected from White and Black patients. Further, these glycans predict opposing overall survival between White and Black patients with prostate cancer. These data suggest differential N-linked glycosylation underline the racial disparity of prostate cancer prognosis. Our study highlights the potential clinical applications of MALDI-MSI for digital pathology and biomarker applications, and reveals molecular features that contribute to the racial disparity in diagnosis and survival of prostate cancer patients. Citation Format: Lindsey R. Conroy, Lyndsay E.A. Young, Grant L. Austin, Alexandra E. Stanback, Derek B. Allison, Matthew S. Gentry, Richard R. Drake, Ramon C. Sun. Mass spectrometry imaging of N-glycans identifies racial discrepancies in human prostate tumors [abstract]. In: Proceedings of the AACR Virtual Special Conference on Tumor Heterogeneity: From Single Cells to Clinical Impact; 2020 Sep 17-18. Philadelphia (PA): AACR; Cancer Res 2020;80(21 Suppl):Abstract nr PO-085.

Molecular signatures of tumor progression in myxoid liposarcoma identified by N-glycan mass spectrometry imaging

AbstractMyxoid liposarcoma (MLS) is the second most common subtype of liposarcoma, accounting for ~6% of all sarcomas. MLS is characterized by a pathognomonic FUS–DDIT3, or rarely EWSR1–DDIT3, gene fusion. The presence of ≥5% hypercellular round cell areas is associated with a worse prognosis for the patient and is considered high grade. The prognostic significance of areas with moderately increased cellularity (intermediate) is currently unknown. Here we have applied matrix-assisted laser desorption/ionization mass spectrometry imaging to analyze the spatial distribution of N-linked glycans on an MLS microarray in order to identify molecular markers for tumor progression. Comparison of the N-glycan profiles revealed that increased relative abundances of high-mannose type glycans were associated with tumor progression. Concomitantly, an increase of the average number of mannoses on high-mannose glycans was observed. Although overall levels of complex-type glycans decreased, an increase of tri- and tetra-antennary N-glycans was observed with morphological tumor progression and increased tumor histological grade. The high abundance of tri-antennary N-glycan species was also associated with poor disease-specific survival. These findings mirror recent observations in colorectal cancer, breast cancer, ovarian cancer, and cholangiocarcinoma, and are in line with a general role of high-mannose glycans and higher-antennary complex-type glycans in cancer progression.This study on myxoid liposarcoma, based on mass spectrometry imaging of N-glycans, has revealed that increases in high-mannose-type as well as tri- and tetra-antennary complex-type N-glycans are associated with morphological progression of the disease. The increased abundance of tri-antennary complex-type glycans was also associated with a poor disease-specific survival for the patients.