Figure 2.  .

Comparative analysis of predicted secondary structure. (A) NetOGlyc 3.1 predicts at least five O-linked glycosylation sites (round knobs) in horse versus 13 in humans. N-linked glycosylation centered on asparagine 119 in human lacritin is predicted by NetNGlyc 1.0 (square knob). PSIPRED (version 3.0) predicts several C-terminal α-helices in horse lacritin, much like human. The two large C-terminal helices in human lacritin have been validated by circular dichroism² (Beck SL, Laurie GW, unpublished data, 2005). The human N-Pep Lac peptide and recombinant truncation mutant N-65 are antigens for anti-N- and anti-C-terminal lacritin antibodies noted in Figure 1. SP, signal peptide. Asterisks indicate regions further analyzed. In human lacritin, the C-terminal α-helix (*) forms the syndecan-1 binding domain for cell targeting.⁸ (B) PONDR analysis of human and horse lacritin sequences. The y-axis indicates predicted disorder and order, respectively, above and below the x-axis. Protein length is represented along the x-axis with the amino terminal signal peptide indicated by the ordered domain at left. The slightly longer human lacritin tracing has been compressed to fit on the same x-axis scale with the horse. PONDR predicts that human lacritin may contain more C-terminal order than horse lacritin. (C) PEPWHEEL analysis of α-helical regions indicated by asterisks in (A). The distinctive hydrophobic face (adjacent boxed residues) of this region in human lacritin² is partially conserved in horse lacritin. The human hydrophobic face is required for syndecan-1 binding, a function predicted to be shared by horse lacritin. Amino acids with nonpolar, basic, acidic, and uncharged side chains are respectively blue, green, brown, and black.