Abstract
AMACO (VWA2 protein) is an extracellular matrix protein of unknown function associated with certain basement membranes in skin, lung, and kidney. AMACO is a member of the von Willebrand factor A-like (VWA) domain containing protein superfamily and in addition to three VWA domains it also contains two epidermal growth factor-like domains. One of these contains the rare, overlapping consensus sequences for both O-glucosylation and O-fucosylation. In earlier studies of other proteins the attachment of either core glucose and fucose moieties or of the respective elongated glycans starting with these monosaccharides has been described. By a detailed mass spectrometric analysis we show that both elongated O-glucosylated (Xyl1-3Xyl1-3Glc) and elongated O-fucosylated glycan chains (NeuAc2-3Gal1-4GlcNAc1-3Fuc) can be attached to AMACO in close proximity on the same epidermal growth factor-like domain. It has been reported that the lack of O-fucosylation can markedly decrease secretion of proteins. However, the secretion of AMACO is not significantly affected when the glycosylation sites are mutated. The number of extracellular matrix proteins carrying the overlapping consensus sequence is very limited and it could be that these modifications have a new, yet unknown function.
Highlights
MATERIALS AND METHODSExpression and Purification of Recombinant AMACO Fragments—AMACO fragments (P1–P3) were generated by PCR on full-length cDNA clones with the following primers: P1 forward, 5Ј-GCT AGC CCC GAC CAT CTC TCT TCA G-3Ј; P1 reverse, 5Ј-GGA TCC GTC TGG ATC AGC AGT GGT G-3Ј; P2 forward, 5Ј-GCT AGC CAC CAC TGC TGA TCC AGA C-3Ј; P2 reverse, 5Ј-GGA TCC TGG CTG GCT GCA TAG CCT C-3Ј; P3 forward, 5Ј-GCT AGC CCA GCC ACG GCC AGG CTG-3Ј; and P3 reverse, 5Ј-GGA TCC CTT GGC GGA GGA CAG GGC-3Ј
Obvious similarity to any other known domain
We show for the first time that both fully elongated O-glucosylated and O-fucosylated glycan chains can occur on the same epidermal growth factor-like (EGF) domain and that extracellular matrix proteins can be so modified
Summary
Expression and Purification of Recombinant AMACO Fragments—AMACO fragments (P1–P3) were generated by PCR on full-length cDNA clones with the following primers: P1 forward, 5Ј-GCT AGC CCC GAC CAT CTC TCT TCA G-3Ј; P1 reverse, 5Ј-GGA TCC GTC TGG ATC AGC AGT GGT G-3Ј; P2 forward, 5Ј-GCT AGC CAC CAC TGC TGA TCC AGA C-3Ј; P2 reverse, 5Ј-GGA TCC TGG CTG GCT GCA TAG CCT C-3Ј; P3 forward, 5Ј-GCT AGC CCA GCC ACG GCC AGG CTG-3Ј; and P3 reverse, 5Ј-GGA TCC CTT GGC GGA GGA CAG GGC-3Ј. The amplified PCR products for the full-length AMACO were cloned in a modified pCEP-Pu vector [17], containing an N-terminal BM-40 signal peptide and a C-terminal 2 ϫ Strep tag. Secretion of recombinant proteins into the cell culture medium was confirmed by SDS-PAGE followed by immunoblotting with specific antisera directed against full-length AMACO and by peptide mass fingerprinting. The samples were diluted 1:4 with 50 mM Tris-HCl, pH 8.0, trypsin (sequencing grade, Promega) added to a final concentration of 12.5 ng/l, followed by incubation at 37 °C overnight. The methylated glycan samples (approximately 500 pmol/l) contained in methanol were applied to the stainless steel target by mixing a 0.5-l aliquot with 1.0 l of matrix (saturated solution of 2,5-dihydroxybenzoic acid in acetonitrile, 0.1% trifluoroacetic acid, 1:2).
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