ABSTRACT
A prominent 45-kDa component was identified by protein staining following SDS-polyacrylamide gel electrophoresis of a 4 M guanidine hydrochloride extract from bovine vitreous collagen fibrils. Peptide sequences obtained from this component were used as a basis for the cloning (from human retinal cDNA) and sequencing of a novel member of the leucine-rich repeat extracellular matrix protein family that we have named opticin. Opticin mRNA was found by reverse transcription polymerase chain reaction in ligament and skin as well as in retina. An open reading frame containing 332 amino acids was identified, the first 19 amino acids representing a signal peptide. The deduced amino acid sequence of the mature protein encodes a 35-kDa protein with a calculated isoelectric point of 5.4. The central domain of this protein consists of six B-type leucine-rich repeats. This domain is flanked by cysteine clusters including a C-terminal two-cysteine cluster containing an additional leucine-rich repeat. The N-terminal region contains a cluster of potential O-glycosylation sites, and analysis of bovine vitreous opticin demonstrated the presence of sialylated O-linked oligosaccharides substituting the core protein. Opticin shows highest protein sequence identity to epiphycan (42%) and osteoglycin (35%) and belongs to Class III of the leucine-rich repeat extracellular matrix protein family.
Subject(s)
Extracellular Matrix Proteins/chemistry , Extracellular Matrix Proteins/genetics , Extracellular Matrix/chemistry , Vitreous Body/chemistry , Amino Acid Sequence , Animals , Base Sequence , Blotting, Northern , Cattle , Collagen/metabolism , Electrophoresis, Polyacrylamide Gel , Extracellular Matrix Proteins/isolation & purification , Glycoproteins/chemistry , Humans , Intercellular Signaling Peptides and Proteins , Leucine/chemistry , Male , Molecular Sequence Data , Multigene Family , Peptides/metabolism , Phylogeny , Proteoglycans/chemistry , Retina/chemistry , Reverse Transcriptase Polymerase Chain Reaction , Sequence Homology, Amino Acid , Small Leucine-Rich Proteoglycans , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Tissue DistributionABSTRACT
Adult and foetal bovine vitreous were analyzed for the presence of unprocessed and partially processed forms of type II procollagen. Type II procollagen can exist in two alternatively spliced forms: a short form which lacks a large part of the N-propeptide due to the splicing out of exon 2 and a long form in which exon 2 is expressed and which has a full-sized N-propeptide. Both splice variants were demonstrated and analyses of type II pN-collagen demonstrated that the long form predominated, the ratio of the long form to the short form being 5:1 in foetal vitreous and 1.5:1 in adult vitreous. In foetal vitreous 35% of the type II collagen extracted was in the pN form, but there was very little unprocessed type II procollagen or type II pC-collagen. In contrast, adult vitreous extracts contained relatively less type II pN-collagen, but contained larger amounts of type II pC-collagen and unprocessed type II procollagen.
