Presence of tear lipocalin and other major proteins in lacrimal fluid of rabbits

Abstract

The lipocalins are a highly divergent, ubiquitous family of proteins that commonly function in binding lipophilic molecules. Although a specific tear lipocalin is a major component of lacrimal fluid and tears in many mammals, there has been no definitive identification of such a protein in rabbit tears. The goals of this project were to identify the major proteins in rabbit ( Oryctolagus cuniculus) lacrimal fluid, so as to determine if they include a lipocalin and, if such a protein is present, to determine its source. Lacrimal fluid was collected from NZW sexually mature female rabbits, and culture medium from rabbit lacrimal gland epithelial (acinar) and interstitial cells was isolated. Proteins from these fluids were separated by SDS-PAGE electrophoresis and analyzed by sequencing the intact proteins and sequencing or mass analysis of fragments derived by trypsin digestion. Proteins of approximately 85 and 67 kDa were identified as rabbit transferrin and serum albumin, respectively, while components of 17 and 7 kDa had N-terminal sequences identical to those of lipophilin CL and AL, respectively. BLAST searches of the nr database with the N-terminal sequence of a protein of 18 kDa did not identify any homologues. However, when used to scan the PROSITE database, it was found to contain a lipocalin signature sequence. It is closely related to two lipocalins previously isolated from rabbit saliva and nasal mucus. Further studies with the N-terminal and internal sequences confirmed that the lacrimal protein is a lipocalin that is truncated at the N-terminus as compared with other tear lipocalins and is more similar to odorant binding proteins from rodents.