Identification of two highly sialylated human tear-fluid DMBT1 isoforms: the major high-molecular-mass glycoproteins in human tears.
Human open eye tear fluid was separated by low-percentage SDS/PAGE to detect high-molecular-mass protein components. Two bands were found with apparent molecular masses of 330 and 270 kDa respectively. By peptide-mass fingerprinting after tryptic digestion, the proteins were found to be isoforms of the DMBT1 gene product, with over 30% of the predicted protein covered by the tryptic peptides. By using gradient SDS/agarose/polyacrylamide composite gel electrophoresis and staining for glycosylation, it was shown that the two isoforms were the major high-molecular-mass glycoproteins of >200 kDa in human tear fluid. Western blotting showed that the proteins expressed sialyl-Le(a). After the release of oligosaccharides by reductive beta-elimination from protein blotted on to PVDF membrane, it was revealed by liquid chromatography-MS that the O-linked oligosaccharides were comprised mainly of highly sialylated oligosaccharides with up to 16 monosaccharide units. A majority of the oligosaccharides could be described by the formula dHex(0-->2)NeuAc(1-->)(x)Hex(x)HexNAc(x)(-ol), x=1-6, where Hex stands for hexose, dHex for deoxyhexose, HexNAc for N-acetylhexosamine and NeuAc for N-acetylneuraminate. The number of sialic acids in the formula is less than 5. Interpretation of collision-induced fragmentation tandem MS confirmed the presence of sialic acid and suggested the presence of previously undescribed structures carrying the sialyl-Le(a) epitopes. Small amounts of neutral and sulphated species were also present. This is the first time that O-linked oligosaccharides have been detected and described from protein variant of the DMBT1 gene.
Schulz
Benjamin L
BL
Proteome Systems Limited, Locked Bag 2073, North Ryde, Sydney, NSW 1670, Australia.
Oxley
David
D
Packer
Nicolle H
NH
Karlsson
Niclas G
NG
D000373:Agglutinins; D000595:Amino Acid Sequence; D002135:Calcium-Binding Proteins; D002240:Carbohydrate Sequence; D002853:Chromatography, Liquid / Q000379:methods; D004268:DNA-Binding Proteins; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D006023:Glycoproteins / Q000737:chemistry* / Q000302:isolation & purification; D006801:Humans; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009844:Oligosaccharides / Q000737:chemistry*; D020033:Protein Isoforms / Q000737:chemistry / Q000302:isolation & purification; D011956:Receptors, Cell Surface / Q000737:chemistry* / Q000302:isolation & purification; D012794:Sialic Acids; D013666:Tears / Q000737:chemistry*; D025521:Tumor Suppressor Proteins
D000373:Agglutinins; D002135:Calcium-Binding Proteins; C108174:DMBT1 protein, human; D004268:DNA-Binding Proteins; D006023:Glycoproteins; D009844:Oligosaccharides; D020033:Protein Isoforms; D011956:Receptors, Cell Surface; D012794:Sialic Acids; D025521:Tumor Suppressor Proteins