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Yinghui Zhang, Ronald W. Raab, Robert L. McKown, Yves Durocher, Gordon W. Laurie; Role Of Syndecan-1 Sulfation In Cell Surface Targeting By Tear Prosecretory Mitogen Lacritin. Invest. Ophthalmol. Vis. Sci. 2011;52(14):3715.
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© ARVO (1962-2015); The Authors (2016-present)
Tear prosecretory factor ‘lacritin’ targets epithelia via a heparanase dependent ‘off/on’ switch. When ‘on’, the lacritin binding sequence GAGAL in ocular surface proteoglycan syndecan-1 (SDC1) is exposed and up to three heparan sulfate stubs are generated. Mutational analysis suggests that stubs at serines 23 and 25, but not 15 contribute to lacritin binding. Here we ask whether stub binding activity derives from predicted 3-O, 2-O and/or N-sulfation sites on HS stub termini. 2-O and 3-O sulfotransferase mRNAs are downregulated in conjunctiva from human non-Sjogren’s dry eye (Mantelli F et al, IOVS ‘09)
SDC1 binding of lacritin was assayed in pull-down assays using lysates from SDC1 stably transfected cells either without or with epitope-specific anti-heparan and chondroitin sulfate antibodies (single chain variable fragment) from Dr. Toin H. Van Kuppevelt. Lysates were also collected from the same cells grown in low sulfate medium supplemented with sulfotransferase inhibitor sodium chlorate, and from other cells expressing SDC1 in which the predicted chondroitin sulfate attachment sites at S108 and S194 had been mutated to alanine.
Sulfate-deficient SDC1 bound lacritin substantially less well than sulfated SDC1. At relatively low concentration, purified antibodies HS4C3 and IO3H10, but consistently not AO4B08 and HS4E4 nor negative control MPB49 inhibited lacritin binding to SDC1. At the highest concentration, AO4B08 and HS4E4, but not MPB49, also inhibited binding. HS4C3 binds most strongly to a rare 3-O sulfated modification, whereas AO4B08 and HS4E4 recognize other heparan sulfate modifications, including 2-O, 6-O and N-sulfation. Surprisingly, IO3H is specific for chondroitin sulfate. SDC1 S108A/S194A appears to bind less well.
Heparanase generates a binding site consisting of HS stubs whose 3-O sulfation (and possibly chondroitin sulfate) appear to contribute to lacritin binding, and may be suboptimal in non-Sjogrens dry eye.
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