Biosynthesized Multivalent Lacritin Peptides Stimulate Exosome Production in Human Corneal Epithelium.

Changrim Lee,Gordon W Laurie,J Andrew Mackay,Sarah F Hamm-Alvarez,Maria C Edman

doi:10.3390/ijms21176157

Abstract

Lacripep is a therapeutic peptide derived from the human tear protein, Lacritin. Lacripep interacts with syndecan-1 and induces mitogenesis upon the removal of heparan sulfates (HS) that are attached at the extracellular domain of syndecan-1. The presence of HS is a prerequisite for the syndecan-1 clustering that stimulates exosome biogenesis and release. Therefore, syndecan-1-mediated mitogenesis versus HS-mediated exosome biogenesis are assumed to be mutually exclusive. This study introduces a biosynthesized fusion between Lacripep and an elastin-like polypeptide named LP-A96, and evaluates its activity on cell motility enhancement versus exosome biogenesis. LP-A96 activates both downstream pathways in a dose-dependent manner. HCE-T cells at high confluence treated with 1 μM LP-A96 enhanced cell motility equipotent to Lacripep. However, cells at low density treated with 1 μM LP-A96 generated a 210-fold higher number of exosomes compared to those treated at low density with Lacripep. As monovalent Lacripep is capable of enhancing cell motility but not exosome biogenesis, activation of exosome biogenesis by LP-A96 not only suggests its utility as a novel molecular tool to study the Lacritin biology in the corneal epithelium but also implies activity as a potential therapeutic peptide that can further improve ocular surface health through the induction of exosomes.

Highlights

The lacrimal gland-corneal axis plays a critical role in maintaining ocular health
‘LP’ refers to the lacritin-derived peptide used to generate LP-A96, and ‘Lacripep’ refers to the peptide equivalent to the one currently in clinical trials (NCT03226444). Both the LP and Lacripep used in this study are derived from the active fragment at the C-terminus of the human Lacritin: GKQFIENGSEFAQKLLKKFSLLKPWA. While both LP and Lacripep share an identical 19 amino acid sequence as a core (Table 1), LP contains five additional amino acids: the N-terminal methionine encoded by the start codon followed by a glycine (MG), which is found in human lacritin
This study describes the construction of a multivalent Lacritin-derived peptide nanoparticle named LP-A96 and its biological effects in corneal epithelial cells

Summary

Introduction

The lacrimal gland-corneal axis plays a critical role in maintaining ocular health. The lacrimal gland is the major organ responsible for the secretion of essential proteins and electrolytes into the tear film that overlays and protects the cornea and conjunctiva [1]. Discovered by the Laurie laboratory [3], several studies have revealed that the active monomeric form of Lacritin is significantly downregulated in patients suffering from chronic blepharitis [4], aqueous-deficient dry eye [5], contact-lens related dry eye [6], and dry eyes associated with primary Sjögren’s syndrome (SS) [7] It remains unclear if Lacritin monomer down-regulation (in part through tissue transglutaminase-dependent cross-linking at the syndecan-1 binding domain [8]) is a symptom or a direct cause of these ocular surface diseases; Lacritin has shown potential as a therapeutic molecule. Its supplementation enhanced tear secretion from rat LGAC [3] and monkey LGAC [9], increased basal tear secretion in rabbit LGAC [10] and dry eye mouse eyes [11], and stimulated human corneal epithelial (HCE-T) cell proliferation [12] This composite of preclinical evidences supports the continued development of Lacritin as an ocular therapeutic. Its peptide derivative, LacripepTM, is under clinical evaluation for protein replacement therapy for dry eye disease (DED) and SS-associated DED (NCT03226444)

Methods

Results

Conclusion