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

Glycosylation of proteins greatly affects their structure and function, but traditional genomics and transcriptomics are not able to precisely capture tissue- or species-specific 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 Galα1–3Gal 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 Galα1–3Gal epitope. These findings provide mechanistic insight into the causal connection between the genotype and the phenotype seen in α3GalT-1-deficient mice and transgenic mice expressing endo-β-galactosidase C. Because humans do not possess the Galα1–3Gal structure on their tissues, we further examined the human dermal and epidermal N-glycome. Comparative glycomics revealed that the GalNAcβ1–4GlcNAc (N,N′-diacetyllactosediamine) epitope, instead of the Galα1–3Gal epitope, was highly expressed in human epidermis. Glycosylation of proteins greatly affects their structure and function, but traditional genomics and transcriptomics are not able to precisely capture tissue- or species-specific 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 Galα1–3Gal 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 Galα1–3Gal epitope. These findings provide mechanistic insight into the causal connection between the genotype and the phenotype seen in α3GalT-1-deficient mice and transgenic mice expressing endo-β-galactosidase C. Because humans do not possess the Galα1–3Gal structure on their tissues, we further examined the human dermal and epidermal N-glycome. Comparative glycomics revealed that the GalNAcβ1–4GlcNAc (N,N′-diacetyllactosediamine) epitope, instead of the Galα1–3Gal 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 (1Ohtsubo K. Marth J.D. Glycosylation in cellular mechanisms of health and disease.Cell. 2006; 126: 855-867Abstract Full Text Full Text PDF PubMed Scopus (1987) Google Scholar). Cells in the epidermis, which forms a major part of the epithelial barrier, undergo desquamation and are continuously being renewed (2Madison K.C. Barrier function of the skin: “la raison d'être” of the epidermis.J. Investig. Dermatol. 2003; 121: 231-241Abstract Full Text Full Text PDF PubMed Scopus (788) Google Scholar), 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 (3Mehul B. Corre C. Capon C. Bernard D. Schmidt R. Carbohydrate expression and modification during keratinocyte differentiation in normal human and reconstructed epidermis.Exp. Dermatol. 2003; 12: 537-545Crossref PubMed Scopus (11) Google Scholar) or monoclonal antibodies (4Dabelsteen E. Buschard K. Hakomori S.-i. Young W.W. Pattern of distribution of blood group antigens on human epidermal cells during maturation.J. Investig. Dermatol. 1984; 82: 13-17Abstract Full Text PDF PubMed Scopus (53) Google Scholar). 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 (5Uematsu R. Furukawa J.-i. Nakagawa H. Shinohara Y. Deguchi K. Monde K. Nishimura S.-I. High throughput quantitative glycomics and glycoform-focused proteomics of murine dermis and epidermis.Mol. Cell. Proteomics. 2005; 4: 1977-1989Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar), 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 glycomics based on the glycoblotting technique (6Nishimura S.-I. Niikura K. Kurogochi M. Matsushita T. Fumoto M. Hinou H. Kamitani R. Nakagawa H. Deguchi K. Miura N. Monde K. Kondo H. High-throughput protein glycomics: combined use of chemoselective glycoblotting and MALDI-TOF/TOF mass spectrometry.Angew. Chem. Int. Ed. Engl. 2005; 44: 91-96Crossref Scopus (130) Google Scholar, 7Furukawa J.-i. Shinohara Y. Kuramoto H. Miura Y. Shimaoka H. Kurogochi M. Nakano M. Nishimura S.-I. A comprehensive approach to structural and functional glycomics based on chemoselective glycoblotting and sequential tag conversion.Anal. Chem. 2008; 80: 1094-1101Crossref PubMed Scopus (119) Google Scholar, 8Miura Y. Hato M. Shinohara Y. Kuramoto H. Furukawa J.-i. Kurogochi M. Shimaoka H. Tada M. Nakanishi K. Ozaki M. Todo S. Nishimura S.-I. BlotGlycoABC™, an integrated glycoblotting technique for rapid and large scale clinical glycomics.Mol. Cell. Proteomics. 2008; 7: 370-377Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). In this method, glycans derived from biological samples are selectively captured onto novel high density hydrazide beads (BlotGlyco H) for highly efficient purification of oligosaccharides from complex biological samples. The captured oligosaccharides are subjected to on-bead methyl esterification to stabilize sialic acid(s) for the simultaneous quantitation of neutral and sialylated oligosaccharides by MALDI-TOF MS and are finally recovered as arbitrary derivatives by imine exchange chemistry. In the current study, we first used this newly established glycoblotting technique to re-examine both the neutral and sialylated N-glycome of murine dermis and epidermis. We detected 75 oligosaccharides, more than twice the amount detected previously when sialic acids were removed, thus enabling us to perform a more detailed comparison of tissue-specific N-glycosylation profiles. We found that the expression of oligosaccharides containing the Galα1–3Gal epitope was only detected in epidermis, although many non-reducing terminal epitopes tend to vanish in epidermis. Tracing from the glycome back to the and allowed us to identify a group of proteins that Galα1–3Gal epitope and a receptor that Galα1–3Gal epitope. The glycans in mice epidermis for the in transgenic mice expressing endo-β-galactosidase C. human epidermal glycomics was to the of a to that of the Galα1–3Gal epitope in murine epidermis. mice were from were a mouse and Full samples were from the of were to the of human samples were from during The of the and of the their of from the the epidermis was from the dermis by for The epidermis and dermis were and for in as by and A rapid of lipid and PubMed Scopus Google Scholar), and were from in previously Y. Miura Y. Furukawa Nakano M. Shinohara Y. M. Nishimura S.-I. glycomics of human glycoproteins based on the for Cell. Proteomics. Full Text Full Text PDF PubMed Scopus Google Scholar) with and tissue to was in in with and with by with used methyl major the samples were with The was to and in of in the were and by using a previously J.-i. Shinohara Y. Kuramoto H. Miura Y. Shimaoka H. Kurogochi M. Nakano M. Nishimura S.-I. A comprehensive approach to structural and functional glycomics based on chemoselective glycoblotting and sequential tag conversion.Anal. Chem. 2008; 80: 1094-1101Crossref PubMed Scopus (119) Google Scholar). of epidermis and dermis samples were to a hydrazide high density (BlotGlyco of in was and the were captured by for the beads were with in and in in was and the was for to the hydrazide the of the beads with and the sialic acids were methyl by for with in The beads were with and and of methyl and of in were The were for to the glycans captured on the beads to and the glycans were with To the an of was to a to the with with the were with in the of the were with and with in was and with of in and an was on the The samples were to the of the different oligosaccharides in MALDI-TOF data were using an mass with a by the of the were using the with an of a of and a of in the The were by the signals of The of mass was by of were by the into the of and was by with by the technique as previously H. Y. K. Y. N. of neutral and from human glycoproteins using of PubMed Scopus Google Scholar, N. Nakagawa H. K. Y. N. of neutral and PubMed Scopus Google Scholar). in the were by and with and of by the were further by the from using The of was to an and the of the were with to the of and by on the were to the the of the on the were by a from these a of and which to of the comparison with the of the from were was by with a on the and sequential of were from the of the were by MS using to the previously M. Nishimura S.-I. of by of MALDI-TOF/TOF mass Chem. PubMed Scopus Google Scholar, M. Nakagawa H. Kurogochi M. Marth J.D. Nishimura S. N-glycan in by a systematic glycomics approach based on and in MALDI-TOF/TOF mass Cell. Proteomics. 2006; Full Text Full Text PDF PubMed Scopus Google Scholar). was as a in The N-glycan samples were in on the of the with of or and were using an mass with a and by the In MALDI-TOF MS by a were to a of by of the were In were to and in a The were further by in the and their were the were by using the and murine epidermis was in a of and with and The proteins were and with The proteins were to a A with a of and the was with the oligosaccharides were with containing methyl The were on an using with a of in was a of and was The was into and with a The were in of A of was by and in the with and was with in and to the MALDI-TOF and MALDI-TOF/TOF data were using an mass as analysis in the were to and in a by of the were further by in the and their were the were by using of MALDI-TOF MS was using and of a of human and and glycoproteins from Dermatol. PubMed Scopus Google Scholar, in during epidermal with lectins and of Scopus Google Scholar, S. 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PubMed Scopus Google Scholar, J.D. the of oligosaccharides of mouse for the receptor S. PubMed Scopus Google Scholar, K. R. with that of mouse to in PubMed Scopus Google Scholar, S. C. K. K. S. T. of acids on and their in cell adhesion to Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, M. H. Hakomori S.-i. of N-glycosylation in or of and and of Chem. Full Text PDF PubMed Google Scholar, S. M. galectin-3 and human epithelial PubMed Google Scholar, H. S. cell PubMed Scopus Google Scholar) were from the using and were by the to based on the a containing mouse The was for with to and a mass for the of and for the of were as being of by was We first the with that their and with signals for or the not contain the for glycosylation the data were of the In sets of data were the of with containing methyl or methyl was further by as previously (5Uematsu R. Furukawa J.-i. Nakagawa H. Shinohara Y. Deguchi K. Monde K. Nishimura S.-I. High throughput quantitative glycomics and glycoform-focused proteomics of murine dermis and epidermis.Mol. Cell. Proteomics. 2005; 4: 1977-1989Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). The quantitation of the of different a N-glycan was by the the from that the of the Galα1–3Gal epitope and with were as previously J.-i. Shinohara Y. Kuramoto H. Miura Y. Shimaoka H. Kurogochi M. Nakano M. Nishimura S.-I. A comprehensive approach to structural and functional glycomics based on chemoselective glycoblotting and sequential tag conversion.Anal. Chem. 2008; 80: 1094-1101Crossref PubMed Scopus (119) Google Scholar). and murine epidermal proteins were with and were subjected to glycoblotting as The oligosaccharides were recovered as which are and for oligosaccharides were subjected to and to the previously R. S. M. K. of in cell of cells using a of and normal 2006; PubMed Scopus Google Scholar). oligosaccharides were in in and were with to the of derivatives to To the an of samples was to an The was onto a murine galectin-3 and were with to carrier were by using a as previously Y. H. M. M. M. Y. M. for oligosaccharides to both and Chem. PubMed Scopus Google Scholar). The cell by a was from the to and We the murine dermal and epidermal using a recently established glycoblotting technique J.-i. Shinohara Y. Kuramoto H. Miura Y. Shimaoka H. Kurogochi M. Nakano M. Nishimura S.-I. A comprehensive approach to structural and functional glycomics based on chemoselective glycoblotting and sequential tag conversion.Anal. Chem. 2008; 80: 1094-1101Crossref PubMed Scopus (119) Google Scholar) that a high density hydrazide (BlotGlyco H) with methyl esterification of sialic acids and tag by a for highly MALDI-TOF in the MALDI-TOF MS from the epidermal glycome to of than dermal We detected 75 oligosaccharides in dermis or epidermis, more than twice the detected previously when sialic acids were (5Uematsu R. Furukawa J.-i. Nakagawa H. Shinohara Y. Deguchi K. Monde K. Nishimura S.-I. High throughput quantitative glycomics and glycoform-focused proteomics of murine dermis and epidermis.Mol. Cell. Proteomics. 2005; 4: 1977-1989Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). This not only to the of a of sialylated but to from the with the glycoblotting technique of high purification The structure and abundance of detected are in We the abundance of of structure in the oligosaccharides High mannose-type oligosaccharides, and were more highly expressed in epidermis than in dermis The abundance of and which are to to the N-glycan M. glycosylation in and PubMed Scopus Google Scholar), was in epidermis than in dermis in with (5Uematsu R. Furukawa J.-i. Nakagawa H. Shinohara Y. Deguchi K. Monde K. Nishimura S.-I. High throughput quantitative glycomics and glycoform-focused proteomics of murine dermis and epidermis.Mol. Cell. Proteomics. 2005; 4: 1977-1989Abstract Full Text Full Text PDF PubMed Scopus (97) Google signals of oligosaccharides from murine in a new of of the murine in were to the of sialic in which neutral glycans were further into high mannose-type glycans and the of of high mannose-type glycans forms and of sialic the of and the of a Galα1–3Gal epitope are as The was based on the and in Although of the signals in have are abundance and which of the of were in epidermis, of dermal were found to sialylated In the sialylated include both and but was the major sialic The abundance of oligosaccharides or more was in epidermis than in than of epidermal oligosaccharides were with of dermal oligosaccharides were The for the epidermal N-glycome may in part to as as This in with the of and their in epidermis, that of epidermal glycoproteins are often by These the epidermal which in of the desquamation of cells and glycoproteins from Dermatol. PubMed Scopus Google Scholar). although many non-reducing terminal epitopes the expression of oligosaccharides containing the Galα1–3Gal epitope was only detected in epidermis The of the Galα1–3Gal epitope in mice epidermis was from the studies using a to to the Galα1–3Gal epitope in during epidermal with lectins and of Scopus Google Scholar), although the approach not the of a or an or The expression profile of Galα1–3Gal in mouse epidermis us to identify proteins that the Galα1–3Gal epitope in murine epidermis. Because the glycans the Galα1–3Gal epitope were oligosaccharides, we used as an for the of the of to which with S. T. of by on PubMed Scopus Google Scholar). were by analysis by the or by the We glycoproteins with N-glycosylation that the Galα1–3Gal epitope in Although we only a of are to the major Galα1–3Gal proteins we with the the glycoproteins as both and are of and are involved in the of proteins and E. The cell from human 2006; PubMed Scopus Google Scholar). a that with the to the which functions as a receptor for a structural in the of epithelial cells in expression of and that Investig. Dermatol. Full Text PDF PubMed Google Scholar). and are of which with S. H. R. expression of an in murine 1984; PubMed Scopus Google Scholar). molecules from the the cell for by cell receptor and adhesion by to adhesion by and PubMed Scopus Google Scholar, of the to with the of a cell Google Scholar, S. C. The between major complex and the of to PubMed Scopus Google Scholar). The functional of not but the in and an extracellular that in cell adhesion molecules and in of this found in extracellular involved in cell adhesion T. a protein in Full Text Full Text PDF PubMed Scopus Google Scholar), of an for this glycoproteins in extracellular and are or of as epitope in a new We further the abundance of different a N-glycan to a previously (5Uematsu R. Furukawa J.-i. Nakagawa H. Shinohara Y. Deguchi K. Monde K. Nishimura S.-I. High throughput quantitative glycomics and glycoform-focused proteomics of murine dermis and epidermis.Mol. Cell. Proteomics. 2005; 4: 1977-1989Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar) glycoproteins as Galα1–3Gal epitope were with oligosaccharides the Galα1–3Gal epitope when the to an was used for the was a of in which oligosaccharides with the Galα1–3Gal epitope were not the major We previously the to with high mannose-type oligosaccharides when a to an was used for the analysis (5Uematsu R. Furukawa J.-i. Nakagawa H. Shinohara Y. Deguchi K. Monde K. Nishimura S.-I. High throughput quantitative glycomics and glycoform-focused proteomics of murine dermis and epidermis.Mol. Cell. Proteomics. 2005; 4: 1977-1989Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). To the of the on the both and to the were in and the of the N-glycan were In to as with the Galα1–3Gal epitope, we previously to with high mannose-type oligosaccharides This analysis revealed that the of from with high mannose-type oligosaccharides, and the of Galα1–3Gal was The was for the of the and proteins. The were with oligosaccharides the Galα1–3Gal epitope. This analysis revealed that the of the different glycosylation on are with oligosaccharides the Galα1–3Gal epitope, with high mannose-type These that the Galα1–3Gal epitope preferentially attached to adhesion and the of this epitope on the protein highly in of M. T. C. for PubMed Scopus Google Scholar). Although been these α3GalT-1-deficient a of the epitope of by D. The for expression in with a of the 2006; PubMed Scopus Google Scholar, of the for that by glycosylation 2003; PubMed Scopus Google Scholar). transgenic mice that endo-β-galactosidase which the terminal Galα1–3Gal were and that the Galα1–3Gal epitope was from and a of mice were to and that in of from the and finally to of the in the S. M. T. T. T. M. and of transgenic mice expressing endo-β-galactosidase 2008; PubMed Scopus Google Scholar). the in the epidermis, the dermis was normal in structure M. S. S. T. T. and differentiation of epidermal keratinocytes in endo-β-galactosidase transgenic 2008; PubMed Scopus Google Scholar). These with the distribution of the Galα1–3Gal epitope in the murine dermis and epidermis in the current as Galα1–3Gal in this study may to the seen of To further these we data with the tissue derived from the amount of expression data in the of the of the Y. T. analysis of the human and mouse S. 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The expression of in epidermis and are or to the thus to the detected in α3GalT-1-deficient mice and transgenic mice expressing endo-β-galactosidase by transcriptomics data We that the Galα1–3Gal epitope adhesion which the seen in transgenic by using the approach to link the glycome to the and The transcriptomics data that and are expressed in the these molecules may involved in the of seen in α3GalT-1-deficient been to the adhesion properties of as the of the and of and receptor S. C. K. K. S. T. of acids on and their in cell adhesion to Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, M. H. Hakomori S.-i. of N-glycosylation in or of and and of Chem. Full Text PDF PubMed Google Scholar). In this the adhesion glycosylation by the Galα1–3Gal epitope in murine epidermis may involved in the of the adhesion properties of proteins. a to this of which epidermal expression more than expression were on the are as are and in and receptor are a group of lectins to to expressed and by of and epithelial cells S. M. galectin-3 and human epithelial PubMed Google Scholar). a of cell in a with cell glycoconjugates H. S. cell PubMed Scopus Google Scholar). This protein an on cell of galectin-3 and S. PubMed Scopus Google Scholar). expression of to that are or are to T. D. M. a of of PubMed Scopus Google Scholar). to a in T. with the process in S. PubMed Scopus Google Scholar). The of have been by many T. Y. N. T. M. T. T. M. 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The of the of N-glycan was by with and are The murine galectin-3 and were the of and and was as changes in the in the of galectin-3 on the structure of the Galα1–3Gal epitopes the in the by an N-glycan with Galα1–3Gal epitope. with with but the was These that galectin-3 to epitope than to we that the or of on the N-glycan with Galα1–3Gal epitopes affects the with the of further the of This may to the of The of been to have a on the of the in the of this of on the of a PubMed Scopus Google Scholar). that N-glycosylation of and to have Galα1–3Gal epitopes are solely by the N-glycan Galα1–3Gal epitopes and the the in of was Although expression do not that these are analysis that galectin-3 preferentially to N-glycan with the Galα1–3Gal epitope than this epitope and thus that the extracellular of cell adhesion proteins and with galectin-3 Galα1–3Gal epitopes on their epidermis. The biological of the of galectin-3 with cell adhesion proteins to further may or as as in the of and cells E. 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Hakomori S.-i. of in by of of and of PubMed Scopus Google Scholar, M. of and in Scopus Google Scholar). we to human epidermal glycomics and To the glycomics of the human dermis and epidermis have not been studied We human dermal and epidermal glycome using the for the murine the MALDI-TOF are in We the structure and abundance of and we the abundance of of structure in the oligosaccharides in The glycomic of both the epidermis and dermis were between mouse and The high mannose-type oligosaccharides and and their were a and and the sialylated and were of abundance in human epidermis and as were for The in dermal glycomic were mostly to the and of human only mouse both and a the signals in the mass of the dermal glycome often by or between human and glycans the Galα1–3Gal epitope were in human dermis or epidermis. we found that the expression of oligosaccharides with the GalNAcβ1–4GlcNAc epitope was high in the human epidermis The of the epitope were based on as as the in MALDI-TOF/TOF mass as in The epitope on the glycoproteins of as but found on glycoproteins PubMed Scopus Google Scholar). In the group of glycoproteins that the epitope and as as and glycoproteins oligosaccharides on and of and sialylated oligosaccharides on and human Chem. Full Text PDF PubMed Google Scholar, and of the for of Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). not this the epitope an in human epidermis as the Galα1–3Gal epitope in murine epidermis. that was that a of with was found by analysis of human and E. K. H. M. of galectin-3 in and and analysis in human and PubMed Scopus Google Scholar). Although detailed the of galectin-3 and proteins been to the epitope may the of these may to that the from the with a and expressed E. K. H. M. of galectin-3 in and and analysis in human and PubMed Scopus Google Scholar). glycomics and of the of the of the epitope in epidermis to In this study, we the murine glycomics and in sialylated based on a recently established glycoblotting technique and MALDI-TOF/TOF MS of the findings was that the Galα1–3Gal epitope highly selectively attached to adhesion molecules of murine epidermis in a glycosylation site-specific manner. These findings provide mechanistic insight into the causal connection between the genotype and the phenotype seen in α3GalT-1-deficient mice and transgenic mice expressing endo-β-galactosidase C. Because protein glycosylation not with the current to the N-glycosylation of proteins on a scale in of the glycosylation and quantitative complex forms of and in with MS of a protein are available C. M. R. of in and Proteomics. 4: PubMed Scopus Google major are to the purification of the of and high throughput protein