Search

The glomerular filtration barrier is composed of podocytes, glomerular basement membrane, and endothelial cells. Disruption of these structures causes several glomerular injuries, such as focal segmental glomerulosclerosis (FSGS). The surface of podocyte apical membranes is coated by negatively charged sialic acids on core 1-derived mucin-type O-glycans. Here, we aimed to investigate the physiological role of core 1-derived O-glycans in the podocytes using adult mice lacking podocyte-specific core 1-derived O-glycans (iPod-Cos). iPod-Cos mice exhibited early and transient proteinuria with foot process effacements and developed typical FSGS-like disease symptoms. To identify the key molecules responsible for the FSGS-like phenotype, we focused on podocalyxin and podoplanin, which possess mucin-type O-glycans. Expression and localization of podocalyxin did not change in iPod-Cos glomeruli. Besides, western blot analysis revealed significantly lower levels of intact podocalyxin in isolated glomeruli of iPod-Cos mice, and high levels of processed forms in iPod-Cos glomeruli, as compared to that in control glomeruli. Conversely, podoplanin mRNA, and protein levels were lower in iPod-Cos mice than in control mice. These results demonstrated that core 1-derived O-glycan on podocytes is required for normal glomerular filtration and may contribute to the stable expression of podocalyxin and podoplanin.

The core 1 structure is the major constituent of mucin-type

For the effective discovery of the biological roles and disease-specific alterations concerning protein glycosylation in tissue samples, it is important to know beforehand the quantitative and qualitative variations of glycan structures expressed in various types of cells, sites, and tissues. To this end, we used laser microdissection-assisted lectin microarray (LMA) to establish a simple and reproducible method for high-throughput and in-depth glycomic profiling of formalin-fixed paraffin-embedded tissue sections. Using this &ldquo, tissue glycome mapping&rdquo, approach, we present 234 glycomic profiling data obtained from nine tissue sections (pancreas, heart, lung, thymus, gallbladder, stomach, small intestine, colon, and skin) of two 8-week-old male C57BL/6J mice. We provided this LMA-based dataset in the similar interface as that of GlycomeAtlas, a previously developed tool for mass spectrometry-based tissue glycomic profiling, allowing easy comparison of the two types of data. This online tool, called &ldquo, LM-GlycomeAtlas&rdquo, allows users to visualize the LMA-based tissue glycomic profiling data associated with the sample information as an atlas. Since the present dataset allows the comparison of glycomic profiles, it will facilitate the evaluation of site- and tissue-specific glycosylation patterns. Taking advantage of its extensibility, this tool will continue to be updated with the expansion of deposited data.

The core 1 β1,3-galactosyltransferase-specific molecular chaperon (Cosmc) is essential for the synthesis of the core 1 structure of mucin-type O-glycans. To clarify the physiological role of core 1-derived O-glycans in macrophages, we exploited the LysM-Cre transgene to generate a conditional Cosmc mutant allele (conditional Cosmc knockout; cKO) in myeloid cells. cKO mice developed normally with no gross phenotypic abnormalities or abnormal peripheral blood counts. Resident peritoneal macrophages (rpMacs) of cKO mice exhibited impaired engulfment of apoptotic cells but showed normal macrophage differentiation and counts. T-cell immunoglobulin and mucin domain-containing molecule 4 (Tim4) is a phosphatidylserine (PS) receptor expressed on rpMacs and possesses a heavily O-glycosylated domain. Tim4 tethers apoptotic cells through PS binding. Expression of the Tim4 transcript was unchanged in cKO rpMacs, whereas flow cytometric, Western and dot blot analyses revealed that Tim4 protein expression in cKO rpMacs was significantly lower than that in wild-type (WT) rpMacs. Moreover, the expression levels of other efferocytosis-related molecules, Mertk, Itgav and Itgb3, were normal in rpMacs. In addition, hypoglycosylated Tim4-FLAG fusion protein sufficiently recognized PS. These results demonstrated that core 1-derived O-glycan is required for Tim4-dependent normal efferocytosis and may contribute to the stable expression of the Tim4 glycoprotein.

The significance of glycomic profiling has been highlighted by recent findings that structural changes of glycans are observed in many diseases, including cancer. Therefore, glycomic profiling of the whole body (glycome mapping) under different physiopathological states may contribute to the discovery of reliable biomarkers with disease-specific alterations. To achieve this, standardization of high-throughput and in-depth analysis of tissue glycome mapping is needed. However, this is a great challenge due to the lack of analytical methodology for glycans on small amounts of endogenous glycoproteins. Here, we established a standardized method of lectin-assisted tissue glycome mapping. Formalin-fixed, paraffin-embedded tissue sections were prepared from brain, liver, kidney, spleen, and testis of two C57BL/6J mice. In total, 190 size-adjusted fragments with different morphology were serially collected from each tissue by laser microdissection and subjected to lectin microarray analysis. The results and subsequent histochemical analysis with selected lectins were highly consistent with previous reports of mass spectrometry-based N- and/or O-glycome analyses and histochemistry. This is the first report to look at both N- and O-glycome profiles of various regions within tissue sections of five different organs. This simple and reproducible mapping approach is also applicable to various disease model mice to facilitate disease-related biomarker discovery.