FIGURE 2.

Lacritin survival activity is defined by C-terminal hydrophobic residues Ile⁹⁸, Phe¹⁰⁴, Leu¹⁰⁸, Leu¹⁰⁹, and Phe¹¹². A, pepwheel analysis of the C-terminal α-helix (amino acids 94–112) of lacritin. Red residues are hydrophobic. B, Predictor of Naturally Disordered Regions (PONDR) tracings of lacritin, lacritin mutants, and splice variant lacritin-c. 0 represents “order,” and 1 represents “disorder” with tracing below the horizontal line predicted to be ordered. The green rectangle approximates the syndecan-1 binding region (17) for cell targeting. C, linear diagrams comparing lacritin point (red ovals) and truncation mutants and splice variant with the novel I3 C terminus (blue). Numbering here and elsewhere excludes the signal peptide. D, HCE-T cells were sensitized overnight with IFNG and then treated for 15 min with different lacritin constructs (10 nm) in the presence of TNF. Viability was assessed by the MTT assay and is expressed as the -fold increase in viability, defined as the ratio of experimental viability to the control viability. Control viability was derived from cells treated with IFNG/TNF alone. Several mutants and the splice variant are not prosurvival (ANOVA with Dunnett's post-test; **, p < 0.01; *, p < 0.05). The viability of F104S, L108/L109/F112S, and lacritin I3-treated cells does not significantly differ from that of IFNG/TNF-stressed cells (ANOVA with Dunnett's post-test, ns, not significant, p > 0.05. E, HCE-T cells were sensitized overnight with IFNG in the presence of no or increasing amounts of xyloside (left panel). The next day, cells were treated for 15 min with 10 nm lacritin in the presence of TNF and xyloside. Viability was assessed by the MTT assay. Other cells were only treated overnight with no or increasing amounts of xyloside, and viability was assessed by the MTT assay (right panel). Xyloside significantly negates lacritin prosurvival activity. It also significantly reduces cell viability in the absence of IFNG/TNF but only slightly (94 ± 1.6 and 93 ± 1.9% versus 100% viability, respectively, for 80 and 90 μm xyloside). Xyloside inhibits heparan and chondroitin sulfate chain attachment and can inhibit hyaluronan synthesis. Left, diagrams of untreated (above) versus xyloside-treated (below) syndecan-1 with heparan and chondroitin sulfate side chains depicted in green and red, respectively. Both are attached to a core protein with a short cytoplasmic domain below the black line. Error bars represent S.E. lacrt, lacritin; SP, signal peptide; xyl, xyloside; aa, amino acids; untreat, untreated.