LARGE Expression Augments the Glycosylation of Glycoproteins in Addition to α-Dystroglycan Conferring Laminin Binding

Mutations in genes encoding glycosyltransferases (and presumed glycosyltransferases) that affect glycosylation and extracellular matrix binding activity of α-dystroglycan (α-DG) cause congenital muscular dystrophies (CMDs) with central nervous system manifestations. Among the identified genes, LARGE is of particular interest because its overexpression rescues glycosylation defects of α-DG in mutations of not only LARGE but also other CMD-causing genes and restores laminin binding activity of α-DG. It is not known whether LARGE protein glycosylates other proteins in addition to α-DG. In this study, we overexpressed LARGE in DG-deficient cells and analyzed glycosylated proteins by Western blot analysis. Surprisingly, overexpression of LARGE in α-DG-deficient cells led to glycosylation dependent IIH6C4 and VIA4-1 immunoreactivity, despite the prevailing view that these antibodies only recognize glycosylated α-DG. Furthermore, the hyperglycosylated proteins in LARGE-overexpressing cells demonstrated the functional capacity to bind the extracellular matrix molecule laminin and promote laminin assembly at the cell surface, an effect that was blocked by IIH6C4 antibodies. These results indicate that overexpression of LARGE catalyzes the glycosylation of at least one other glycoprotein in addition to α-DG, and that this glycosylation(s) promotes laminin binding activity

Structural analysis of carrageenan from farmed varieties of Philippine seaweed

Kappaphycus alvarezii, Eucheuma denticulatum and, more recently, Kappaphycus sp. ‘Sacol’ variety, are the carrageenan-containing red seaweeds currently farmed in the Philippines. The Kappaphycus sp. ‘Sacol’ variety is of particular interest to the Philippine seaweed industry because of its improved resistance to ‘ice-ice’ disease and its fast growing characteristics. Here we report on the detailed chemical analysis of the carrageenan from this species. The native and alkali-modified carrageenans from Kappaphycus sp. ‘Sacol’ variety were characterized by FT-IR, 1H and 13C NMR spectroscopy, and constituent sugar and methylation analyses and were compared to those from K. alvarezii, K. cottonii and Eucheuma denticulatum. The three Kappaphycus species contained predominantly κ-carrageenan with low levels of ι-carrageenan, methylated carrageenan and μ-precursor residues, while Eucheuma denticulatum contained predominantly ι-carrageenan with significant amounts of ν-precursor residues. Taxonomic classification of Kappaphycus species based on morphology has proved to be difficult because of their known plasticity. Molecular analysis using the rbcLsequence revealed that the Kappaphycus sp. ‘Sacol’ variety is most likely a form of K. cottonii

Structural analysis of κ-carrageenan sulfated oligosaccharides by positive mode Nano-ESI-FTICR-MS and MS/MS by SORI-CID

Structural analysis of sulfated oligosaccharides from κ-carrageenan of up to ten residues (MW \u3e2 kDa) was successfully carried out by positive mode nano-ESI-FTICR-MS together with MS/MS using sustained off-resonance irradiation-collision induced dissociation (SORI-CID). Glycosidic bond cleavage reactions via the B- and Y-types of fragmentation were observed and enabled complete sequencing of the oligosaccharide samples. The positions of the labile sulfate substituents were observable using SORI-CID, enabling the determination of the sequence of the sulfated residues