Abstract
Purpose :
Dry Eye Disease affecting 7- 10% of the world’s population is characterized by loss of ocular surface homeostasis yielding chronic inflammation. Lacritin is a prosecretory and regenerative tear (and apparently plasma and CSF) glycoprotein necessary for homeostasis and yet lacking in dry eye. Efficacy was recently validated in a 204-patient phase 2 clinical trial in Primary Sjögren's Syndrome dry eye. We also identified cornea-expressed 'G protein-coupled receptor 87' (GPR87) as the lacritin signaling receptor out of a genome-wide CRISPR/Cas9 death screen using C-terminal lacritin synthetic peptide N-94 as agonist and determined that GPR87 forms an agonist- independent complex with the cell surface heparan sulfate proteoglycan syndecan-1 (SDC1) - an interaction requiring glycosaminoglycan and mutual cytoplasmic domains. Here we further dissect the complex using mutation and peptide inhibition approaches.
Methods :
Eleven different synthetic peptides corresponding to or together spanning each of GPR87's extracellular or intracellular strands or loops were introduced as soluble inhibitors at 20, 100 and 400 μM. Binding studies were performed out of GPR87 and SDC1 expressing cells treated with 25-, 50- or 100-mM chlorate to suppress sulfation, or with glycosaminoglycan assembly inhibitor 4-methylumbelliferyl-b-D-xylopyranoside (xyloside) at 0.01, 0.1 or 1 mM. SDC1 lacking 20 ('del 1-21'), 30 ('1-31') or 200 ('del 51-251') N-terminal amino acids was introduced to GPR87.
Results :
Outer loop peptides - particularly outer loop 3, but no others, inhibited lacritin-GPR87 targeting. Chlorate and xyloside treatment each inhibited SDC1 - GPR87 coupling, in keeping with lack of binding when heparan sulfate could not be substituted at S15, nor chondroitin sulfate at S184 and S185, and new data pointing to the importance of elements within SDC1 amino acids 1 - 21, 1 - 31 and 51 - 252.
Conclusions :
Lacritin targets the outer loop domains of GPR87 (particularly loop 3) as part of a preformed SDC1-GPR87 complex - the latter linked by glycosaminoglycans and by cytoplasmic SDC1 (PDZ binding) and GPR87 ('DRY') domains to regulate ocular surface health.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.