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 270kDa 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 >200kDa in human tear fluid. Western blotting showed that the proteins expressed sialyl-Lea. After the release of oligosaccharides by reductive β-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 dHex0→2NeuAc1→xHexxHexNAcx(-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-Lea 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.
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September 2002
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Research Article|
September 01 2002
Identification of two highly sialylated human tear-fluid DMBT1 isoforms: the major high-molecular-mass glycoproteins in human tears
Benjamin L. SCHULZ;
Benjamin L. SCHULZ
Proteome Systems Limited, Locked Bag 2073, North Ryde, Sydney, NSW 1670, Australia
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David OXLEY;
David OXLEY
Proteome Systems Limited, Locked Bag 2073, North Ryde, Sydney, NSW 1670, Australia
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Nicolle H. PACKER;
Nicolle H. PACKER
Proteome Systems Limited, Locked Bag 2073, North Ryde, Sydney, NSW 1670, Australia
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Niclas G. KARLSSON
Niclas G. KARLSSON
1
Proteome Systems Limited, Locked Bag 2073, North Ryde, Sydney, NSW 1670, Australia
1To whom correspondence should be addressed (e-mail niclas.karlsson@proteomesystems.com).
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Publisher: Portland Press Ltd
Received:
December 21 2001
Revision Received:
April 02 2002
Accepted:
May 17 2002
Accepted Manuscript online:
May 17 2002
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London ©2002
2002
Biochem J (2002) 366 (2): 511–520.
Article history
Received:
December 21 2001
Revision Received:
April 02 2002
Accepted:
May 17 2002
Accepted Manuscript online:
May 17 2002
Citation
Benjamin L. SCHULZ, David OXLEY, Nicolle H. PACKER, Niclas G. KARLSSON; Identification of two highly sialylated human tear-fluid DMBT1 isoforms: the major high-molecular-mass glycoproteins in human tears. Biochem J 1 September 2002; 366 (2): 511–520. doi: https://doi.org/10.1042/bj20011876
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