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Y. Zhang, R. W. Raab, R. L. McKown, Y. Durocher, G. W. Laurie; Prosecretory Mitogen Lacritin Binds Co-Receptor Syndecan-1(SDC1) via a Hybrid Binding Motif of Heparanase-Generated Heparan Sulfate(HS) Stubs and Protein Sequence GAGAL. Invest. Ophthalmol. Vis. Sci. 2010;51(13):4179.
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Lacritin is a 12.3 kD prosecretory mitogen in human tears whose C-terminus targets an unidentified site within the N-terminal 50 aa of SDC1 via a heparanase-dependent off/on switch mechanism (Ma et al, JCB ‘06). It is still not clear whether SDC1’s HS chains sterically block core protein binding or whether the heparanase generated HS stubs contribute to the binding motif. Here we address this question using a new series of SDC1 constructs, synthetic peptides and HS inhibition.
A series of human SDC1 N-terminal truncation, swapping and single or double point mutants were expressed from suspension cultured 293-6E cells and assayed in pulldown experiments.
A) Suspension culture significantly improved SDC1 glycanation, presumably on serines 14, 23 and 24. B) SDC1 truncation of 30, but not 20 aa, and synthetic peptides corresponding to aa 20-30 and 10-30, but not 1-20 and 30-50 abrogated binding to lacritin. Binding affinity was significantly diminished by switching out GAGAL for equivalent SDC2 or SDC4 sequences. Together these data point to a site between aa 20 and 30 whose specificity is designated by the unique SDC1 sequence GAGAL. C) SDC1 point mutant S14A (lacks HS at serine 14) bound lacritin, but not S23/24A (lacking HS at serines 23 and 24). No binding was observed to SDC1 lacking all HS chains (after xyloside treatment). These data rule out steric hindrance, and are in keeping with a role for HS stubs in the GAGAL binding motif. D) Truncation of up to 75 aa from lacritin’s N-terminus did not affect binding. Point mutagenesis of each hydrophobic aa in lacritin’s hydrophobic binding face suggest that leucine 108 and 109 likely interact with elements of SDC1’s hydrophobic GAGAL.
Hydrophobic leucines 108 and 109 of lacritin’s C-terminal amphipathic alpha helix targets a hybrid SDC1 binding site that appears to consist of heparanase-generated HS stubs and SDC1 specific GAGAL. Downregulation of HS glycosyltransferases in dry eye (Mantelli and Argueso, IOVS ’09) would likely affect HS stub composition.
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