Application of cellular O-linked glycomics analysis for the diagnosis of protein glycosylation disorders. M. He1,2, X. Li1,2, M. Raihan1,2, L. Tan1,2, M. Bennett1,2, W. Gahl3, M. Davids3, M. Kane3, C. F. Boerkoel3 1) Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, PA; 2) Palmieri Metabolic Disease Laboratory, Children's Hospital of Philadelphia, Philadelphia, PA; 3) Undiagnosed Disease Program, NHGRI, NIH, Bethesda, MD.

   The glycosylation of protein is an important post-translational modification in many biological systems. Our laboratory has developed the plasma O-glycan assay for diagnosis of the multiple glycosylation disorder, congenital disorder of glycosylation (CDG) type II, using mass spectrometry technology. However, O-linked glycoproteins in peripheral blood only represent a small fraction of O-linked glycoproteome in humans and O-glycan species released from total glycoproteins in plasma are very limited with minimum amount of the core 2 species. Furthermore, it is known that alteration in glycan structure often shortens the half-life of glycoproteins in circulation and the liver plays an active role in removing truncated glycans from the peripheral blood through glycoprotein scavenge pathway. Thus O-linked glycan analysis in plasma only is likely inadequate for detecting defects in O-linked protein glycosylation. In this study, we describe cellular O-glycomics analysis in cultured fibroblast and its application to detect O-linked glycosylation disorders. We found that a board spectra of O-linked GalNAc glycosylation were detected after they were released from fibroblast total glycoproteins using the beta elimination method. The quantification of both mucin core 1 and core 2 species allows measurement of defects in multiple steps in O-linked glycan biosynthesis pathway in fibroblast lines from known CDG patients including GALNT3-CDG, COG7-CDG as well as deficiencies in nucleotide sugar synthesis and transport. We have measured fibroblast O-glycomics in 33 fibroblast lines from patients with borderline changes of O-linked protein glycosylation in plasma. 4 of them (12%) showed profound deficiency in O-linked protein glycosylation that would not be possible to pinpoint in plasma based analysis. Among 43 cell lines from patients with other indications of possible CDG based on unusual N-linked glycan or free glycan profiles of body fluids or clinical findings, 13 of them (30%) showed O-glycomics changes that were significantly deviated from the control group. Our study demonstrates that cellular O-linked glycomics analysis is more informative and sensitive for detecting O-linked or multiple glycosylation disorders comparing to plasma O-glycan analysis alone. Therefore it is an important diagnostic tool for this largely under-recognized group of CDGs.

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