Glycosylation Specific for Adhesion Molecules in Epidermis and Its Receptor Revealed by Glycoform-focused Reverse Genomics

Abstract

Glycosylation of proteins greatly affects their structure and function, but traditional genomics and transcriptomics are not able to precisely capture tissue-or speciesspecific glycosylation patterns. We describe here a novel approach to link different "omics" data based on exhaustive quantitative glycomics of murine dermis and epidermis. We first examined the dermal and epidermal N-glycome of mouse by a recently established glycoblotting technique. We found that the Gal␣1-3Gal epitope was solely expressed in epidermis tissue and was preferentially attached to adhesion molecules in a glycosylation site-specific manner. Clarified glycomic and protemic information combined with publicly available microarray data sets allowed us to identify galectin-3 as a receptor of Gal␣1-3Gal epitope. These findings provide mechanistic insight into the causal connection between the genotype and the phenotype seen in ␣3GalT-1-deficient mice and transgenic mice expressing endo-␤-galactosidase C. Because humans do not possess the Gal␣1-3Gal structure on their tissues, we further examined the human dermal and epidermal N-glycome. Comparative glycomics revealed that the GalNAc␤1-4GlcNAc (N,N-diacetyllactosediamine) epitope, instead of the Gal␣1-3Gal epitope, was highly expressed in human epidermis. Posttranslational protein glycosylation changes the biological and physical properties of glycoconjugates, which include functions as signals or ligands to control their distribution, antigenicity, metabolic fate, stability, and solubility (1). Cells in the epidermis, which forms a major part of the epithelial barrier, undergo desquamation and are continuously being renewed (2), a process that requires changes in adhesion. Because glycoproteins are often involved in adhesion between cells and their extracellular matrices, the glycoproteome of the epidermis may therefore provide new insight into the functional roles of protein glycosylation. Mammalian epidermal glycoconjugates have mostly been studied histochemically using lectins (3) or monoclonal antibodies (4). Although these studies revealed that the cell surfaces of keratinocytes in the epidermis contain numerous glycoconjugates, these approaches cannot provide detailed structural information about the oligosaccharides or their carrier proteins.We previously clarified the quantitative glycomic profile of murine dermis and epidermis using novel aminooxy-based isotope tags and MALDI-TOF MS analysis (5), which revealed distinct features of the N-glycosylation profile of dermis and epidermis. We found that murine epidermal glycoproteins have a high abundance of high mannose-type oligosaccharides, and the striking roles of lysosomal enzymes in epidermis during lipid remodeling and desquamation were further discussed. This study was the first to demonstrate the usefulness of quantitative gross N-glycan profiling for performing systematic glycoform-focused proteomics. To advance and accelerate this approach, we recently established a novel technology platform for large scale quantitative glyc...

Glycan distribution and density in native skin's stratum corneum

“…Corneodesmosomes are major components of intercellular junctions between corneocytes in SC. Some of the key components of corneodesmosomes desmoglein 1, desmocolin 1, and corneodesmosin are N‐linked glycosylatyed proteins . Thus, during the desquamation process, corneodesmosomes are gradually degraded by a sequential action of glycosidases and proteases.…”

Mammalian plasma membrane proteins as potential biomarkers and drug targets

“…protein-protein interaction, PTMs or by simple binding of small ligands, and these molecules can be also a potential drug target. The role of proteomics, especially of plasma membrane proteomics can be: (i) identification of new drug targets, (ii) investigation of the mechanism of actions of the existing ones, (iii) elucidation of the development of drug resistance and (iv) early detection of possible harmful side effects such as immunological reaction of protein drugs [149] or rejection in organ transplantation [79].…”

“…Changes in glycosylation also provide markers for stages in tissue development [77], for cell signaling events [78] as well as for early recognition of potentially harmful reactions after organ transplantation [79]. Methods have been developed for detection of glycosylated membrane proteins and protein fragments in body fluids [80][81][82].…”

Recent Advances in Cellular Glycomic Analyses

“…The blotted glycans were recovered in their reducing terminal-derivative form by adding the aminooxy-containing compound aoWR, which substantially improves detection sensitivity with MALDI-TOF MS analysis [33]. This procedure can be performed in a 96-well plate format and can be used with MALDI-TOF MS for N -glycomic analysis of murine dermis and epidermis [34], as well as murine ES cells and their differentiated progenies ( i.e. , cardiomyocytes or neural cells) [35].…”