Purpose
Lacritin is a small tear protein (~18 kDa) with mitogenic, cytoprotective and prosecretory activities. When added topically to rabbit eyes, lacritin promotes basal tearing. Lacritin targets the corneal epithelial cell surface protein syndecan-1 (SDC1), a widely expressed heparan sulfate proteoglycan via a heparanase dependent mechanism that facilitates interaction between lacritin amino acids 100-109 in its C-terminal alpha-helix and the SDC1 core protein sequence GAGAL nestled in SDC1’s heparan sulfate rich N-terminus. Since GAGAL is hydrophobic, we wondered whether it might influence development of lacritin’s C-terminal alpha helix necessary for binding. Here we performed comparative circular dichroism analyses of lacritin peptide 1898 (aa 95-119) alone, and in the presence of SDC1 peptide 1 (aa 20 - 30), scrambled SDC1 peptide 1, or the equivalent region in SDC4 (SDC4 peptide).
Methods
Synthetic peptides were generated by Genescript to a purity of >95%, dissolved in 10mM dodecylphosphocholine and subjected to CD analysis in the 190-250 nm range with 0.1 nm pitch, 50 nm/min scan speed, 8 second response time and a 2 nm bandwidth. A 1 mm pathlength quartz cuvette with cylindrical geometery (Hellma) was used, stored in 2% Hellmanex prior to usage, washed vigorously 3 times with ddH2O, subsequently washed with 100% ethanol, and then dried completely with pure N2 before and between samples. The spectra displayed represent the average of at least 5 sample runs. Between sample runs, the dd H2O blank was monitored to ensure protein buildup in the cuvette was not occurring.
Results
Lacritin amino acids 95-119 (81 µM) assumed an alpha helical structure in dodecylphosphocholine. This was enhanced by SDC1 peptide 1 (57 µM), but not by scrambled SDC1 peptide 1. SDC4 peptide was partially effective, but less so than SDC1 peptide 1 - in keeping with less hydrophobicity and no interaction of lacritin with SDC4. Alone, SDC1 peptide1, scrambled SDC1 peptide 1 and SDC4 peptide assumed a random coil structure.
Conclusions
Ligation of lacritin’s C-terminal amphiphathic alpha helix with GAGAL in SDC1 is enhanced by mutual hydrophobicity that improves alpha helicity.
Keywords: 486 cornea: tears/tear film/dry eye •
659 protein structure/function •
480 cornea: basic science