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P. Ma, S. Beck, R. Raab, R. McKown, G. Coffman, A. Rapraeger, G.W. Laurie; Identification of Syndecan–1 as a Coreceptor for Prosecretory Mitogen ‘Lacritin’ . Invest. Ophthalmol. Vis. Sci. 2005;46(13):4405.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Lacritin is a small glycoprotein secreted by human lacrimal acinar cells. Lacritin acts in an autocrine or paracrine manner at low nanomolar levels to respectively promote further lacrimal secretion and ductal proliferation. How lacritin targets cells is unknown. Here report on lacritin affinity purification of several cell surface proteins and identification of syndecan–1 as a putative lacritin co–receptor. Methods: Biotinylated cell surface proteins were applied to lacritin affinity columns. After washing extensively in physiological salt–containing buffer, bound protein was eluted with 1 M NaCl, and identified with avidin–peroxidase using an ECL blot method. In controls, columns lacking lacritin were used. Human syndecan–1 plasmid DNA was stably transfected into lacritin unresponsive HEK293 cells. 293T cells stably transfected with human syndecan–2 or –4 were kindly provided by Dr. Atsushi Utani (Chiba University). A series of lacritin C–terminal and N–terminal deletion mutants were generated. Pull down binding assays involved incubation of intein–tagged lacritin or intein deletion mutants with lysates of untransfected or transfected cells followed by precipitation and SDS PAGE. Immunolocalization of syndecan–1 in human lacrimal glands was performed using anti–human syndecan–1 antibody (CD138). Results: The first of approximately four bands repeatedly eluted from lacritin affinity column was determined by sequencing to be syndecan–1. In pull–down assays, lacritin bound mammalian syndecan–1, but not syndecan–2 or 4. The binding appeared to be independent of heparan sulfate side chains, in keeping with lack of lacritin binding to heparin – as previously observed (Sanghi et al, JMB ’01). Deletion of lacritin’s C–terminal 25 but not N–terminal 5,10, or 24 amino acids eliminated binding. Soluble lacritin and N–terminal 24 deletion mutant but not heparin and C–terminal deletion mutants inhibited binding in competitive binding assays. Syndecan–1 was detected in ‘near apical’ and basolateral plasma membranes in human lacrimal gland. Conclusions: Syndecan–1, a coreceptor for FGF’s, Wnt’s and other growth factors appears to serve in the same capacity for lacritin, but in a unique manner apparently independent of heparan sulfate side chains. Dependence on lacritin’s C–terminal 25 amino acids for binding, as well as mitogenesis and calcium signaling, suggests involvement of lacritin’s C–terminal amphipathic α–helix in lacritin–receptor interactions.
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