Mouse tissue glycome atlas 2022 highlights inter-organ variation in major N-glycan profiles.

This study presents "mouse tissue glycome atlas" representing the profiles of major N-glycans of mouse glycoproteins that may define their essential functions in the surface glycocalyx of mouse organs/tissues and serum-derived extracellular vesicles (exosomes). Cell surface glycocalyx composed of a variety of N-glycans attached covalently to the membrane proteins, notably characteristic "N-glycosylation patterns" of the glycocalyx, plays a critical role for the regulation of cell differentiation, cell adhesion, homeostatic immune response, and biodistribution of secreted exosomes. Given that the integrity of cell surface glycocalyx correlates significantly with maintenance of the cellular morphology and homeostatic immune functions, dynamic alterations of N-glycosylation patterns in the normal glycocalyx caused by cellular abnormalities may serve as highly sensitive and promising biomarkers. Although it is believed that inter-organs variations in N-glycosylation patterns exist, information of the glycan diversity in mouse organs/tissues remains to be elusive. Here we communicate for the first-time N-glycosylation patterns of 16 mouse organs/tissues, serum, and serum-derived exosomes of Slc:ddY mice using an established solid-phase glycoblotting platform for the rapid, easy, and high throughput MALDI-TOFMS-based quantitative glycomics. The present results elicited occurrence of the organ/tissue-characteristic N-glycosylation patterns that can be discriminated to each other. Basic machine learning analysis using this N-glycome dataset enabled classification between 16 mouse organs/tissues with the highest F1 score (69.7-100%) when neural network algorithm was used. A preliminary examination demonstrated that machine learning analysis of mouse lung N-glycome dataset by random forest algorithm allows for the discrimination of lungs among the different mouse strains such as the outbred mouse Slc:ddY, inbred mouse DBA/2Crslc, and systemic lupus erythematosus model mouse MRL-lpr/lpr with the highest F1 score (74.5-83.8%). Our results strongly implicate importance of "human organ/tissue glycome atlas" for understanding the crucial and diversified roles of glycocalyx determined by the organ/tissue-characteristic N-glycosylation patterns and the discovery research for N-glycome-based disease-specific biomarkers and therapeutic targets.

Selective reaction monitoring approach using structure-defined synthetic glycopeptides for validating glycopeptide biomarkers pre-determined by bottom-up glycoproteomics.

Clusterin is a heavily glycosylated protein that is upregulated in various cancer and neurological diseases. The findings by the Hancock and Iliopoulos group that levels of the tryptic glycopeptide derived from plasma clusterin, 372Leu-Ala-Asn-Leu-Thr-Gln-Gly-Glu-Asp-Gln-Tyr-Tyr-Leu-Arg385 with a biantennary disialyl N-glycan (A2G2S2 or FA2G2S2) at Asn374 differed significantly prior to and after curative nephrectomy for clear cell renal cell carcinoma (RCC) patients motivated us to verify the feasibility of this glycopeptide as a novel biomarker of RCC. To determine the precise N-glycan structure attached to Asn374, whether A2G2S2 is composed of the Neu5Acα2,3Gal or/and the Neu5Acα2,6Gal moiety, we synthesized key glycopeptides having one of the two putative isomers. Selective reaction monitoring assay using synthetic glycopeptides as calibration standards allowed "top-down glycopeptidomics" for the absolute quantitation of targeted label-free glycopeptides in a range from 313.3 to 697.5 nM in the complex tryptic digests derived from serum samples of RCC patients and healthy controls. Our results provided evidence that the Asn374 residue of human clusterin is modified dominantly with the Neu5Acα2,6Gal structure and the levels of clusterin bearing an A2G2S2 with homo Neu5Acα2,6Gal terminals at Asn374 decrease significantly in RCC patients as compared with healthy controls. The present study elicits that a new strategy integrating the bottom-up glycoproteomics with top-down glycopeptidomics using structure-defined synthetic glycopeptides enables the confident identification and quantitation of the glycopeptide targets pre-determined by the existing methods for intact glycopeptide profiling.

Antiadhesive nanosome elicits role of glycocalyx of tumor cell-derived exosomes in the organotropic cancer metastasis.

Despite emerging importance of tumor cells-derived exosomes in cancer metastasis, the heterogeneity of exosome populations has largely hampered systemic characterization of their molecular composition, biogenesis, and functions. This study communicates a novel method for predicting and targeting pre-metastatic sites based on an exosome model "fluorescent cancer glyconanosomes" displaying N-glycans of cultured tumor cells. Glycoblotting by antiadhesive quantum dots provides a nice tool to shed light on the pivotal functions of the glycocalyx reconstructed from four cancer cell types without bias due to other compositions of exosomes. In vivo imaging revealed that circulation, clearance, and organotropic biodistribution of cancer glyconanosomes in mice depend strongly on cancer cell-type-specific N-glycosylation patterns, the compositions of key glycotypes, particularly dominant abundances of high mannose-type N-glycans and the position-specific sialylation. Notably, organ biodistribution of cancer glyconanosomes is reproducible artificially by mimicking cancer cell-type-specific N-glycosylation patterns, demonstrating that nanosomal glycoblotting method serves as promising tools for predicting and targeting pre-metastatic sites determined by the glycocalyx of extracellular vesicles disseminated from the primary cancer site.

Analysis of the correlation between alterations in N­glycans and invasiveness in liver cancer cell lines.

The N­glycoforms of glycoproteins modify protein function and control a number of biological pathways. The aim of the present study was to investigate the correlation between alterations in N­glycans and cancer aggressiveness in terms of cancer cell invasion ability. The expression of urokinase­type plasminogen activator (uPA) and N­acetylglucosaminyltransferase V (GnT­V) in liver cancer cell lines was analyzed by western blotting. Cell invasiveness was analyzed by Matrigel invasion assays. uPA and GnT­V expression in liver cancer cell lines was knocked down by RNA interference. Furthermore, uPA was overexpressed in liver cancer cells using lentiviral vectors, and a mutant strain of HepG2 cells overexpressing uPA deficient in N­glycans was established. A glycoblotting­assisted matrix­assisted laser desorption/ionization­time­of­flight/mass spectrometry­based quantitative analysis of liver cancer cell lines was performed, in which invasiveness was altered by modifying the expression of uPA and GnT­V. N­glycan profiles were found to differ between the highly invasive liver cancer cell line HLE and the less invasive cell line HepG2. The expression of several N­glycans, including a form with m/z=1892, was changed according to invasiveness controlled by knockdown and overexpression of uPA. The invasiveness of HepG2 cells with mutant uPA did not increase regardless of the level of expression of uPA. Following GnT­V knockdown and N­glycan alteration, uPA expression did not change, whereas cell invasiveness decreased. One N­glycan (m/z=1892) was common among N­glycans in the comparative analysis between HLE and HepG2, HLE and uPA knockdown HLE, HepG2 and uPA­overexpressing HepG2, and HLE and GnT­V knockdown HLE cells and among N­glycan profiles in human uPA. Therefore, N­glycosylation is an important factor controlling invasiveness of liver cancer cells, and a specific N­glycan (m/z=1892) associated with the invasion of liver cancer cells via uPA was identified in the present study.