Lacritin is a pluripotent tear protein that promotes basal tearing,
3 is cytoprotective against inflammatory cytokines, and promotes the proliferation of subconfluent human corneal epithelial cells.
5 Although lacritin purified from monkey tears is prosecretory,
15 all of lacritin's known activities were discovered with recombinant human lacritin generated by
E. coli , historically the most efficient protein manufacturing approach. Current yields are sufficient for research, but not for scale up. Problems could derive from hydrophobic, acidic, or basic residues. For example, hydrophobic residues can promote misfolding. Misfolded proteins aggregate and become secluded in inclusion bodies.
16 Secreted lacritin has 61 residues with nonpolar side chains (
Fig. 1A; hydrophobic, magenta; very hydrophobic, purple), including 34 of 59 residues in the C-terminal half where all known lacritin activities have been attributed.
2 Of these, 19 (of 22 for all of lacritin) are highly hydrophobic. We mutated 11 to serine. Lacritin A105S was insoluble. However, lacritins I68S, I68S/I78S, V69S, I73S, V91S, I98S, F104S, L108S, L109S, and F112S were completely soluble and were generated with yields two or three times that of unaltered lacritin (
Fig. 1B). Lacritin protects human corneal epithelial cells that have been stressed with the inflammatory cytokines interferon-γ and tumor necrosis factor (Wang N, et al.
IOVS 2011;52:ARVO E-Abstract 3712), and requires syndecan-1 for cell targeting.
8 Lacritins I98S, F104S, and F112S were inactive in cytoprotection assays (Wang N, et al.
IOVS 2011;52:ARVO E-Abstract 3712), and I73S, L108S, L109S, and F112S failed to bind syndecan-1 (Zhang Y, et al.
IOVS 2010;51:ARVO E-Abstract 4179). However, lacritins V69S, I73S, L108S, and L109S each were fully cytoprotective (Wang N, et al.
IOVS 2011;52:ARVO E-Abstract 3712), and lacritins I68S, V69S, V91S, I98S, and F104S fully bound syndecan-1 (Zhang Y, et al.
IOVS 2010;51:ARVO E-Abstract 4179).