Characterization of opticin and evidence of stable dimerization in solution.
Opticin is a class III member of the extracellular matrix small leucine-rich repeat protein (SLRP) family that was initially identified in the eye in association with the collagen fibrils of the vitreous humor. Recombinant and tissue-extracted forms of bovine opticin were subjected to biochemical and biophysical characterization. Following SDS-PAGE the predominant component produced by both forms was a broad band between 45-52 kDa. There was evidence for two-stage processing and, additionally, a proteolytic cleavage product of approximately 25 kDa. Deconvolution of circular dichroism spectra revealed beta-sheet (41%), beta-turn (21%), and alpha-helix (10%), and thermal denaturation experiments showed a transition with a midpoint of 47 degrees C. Weight-averaged molecular mass measurements using both light scattering and analytical ultracentrifugation demonstrated that opticin exists in solution as a stable dimer of approximately 90 kDa, which can be dissociated into a monomer by denaturation with 2.5 m guanidine hydrochloride or during SDS-polyacrylamide electrophoresis. Opticin remains a dimer after removal of the amino-terminal region by O-sialoglycoprotein endopeptidase digestion, suggesting that dimer formation is mediated by the leucine-rich repeats. Dimerization could have a number of functional consequences, including divalent ligand interactions.
Le Goff
Magali M
MM
Wellcome Trust Centre for Cell-Matrix Research, School of Biological Sciences and Research Group in Eye & Vision Science, The Medical School, University of Manchester, Manchester M13 9PT, United Kingdom.
Hindson
Vincent J
VJ
Jowitt
Thomas A
TA
Scott
Paul G
PG
Bishop
Paul N
PN
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002417:Cattle; D002850:Chromatography, Gel; D002942:Circular Dichroism; D003001:Cloning, Molecular; D003094:Collagen / Q000378:metabolism; D019281:Dimerization; D004285:Dogs; D004591:Electrophoresis, Polyacrylamide Gel; D016326:Extracellular Matrix Proteins / Q000737:chemistry* / Q000302:isolation & purification*; D005822:Genetic Vectors; D019791:Guanidine / Q000494:pharmacology; D006801:Humans; D008024:Ligands; D008027:Light; D008969:Molecular Sequence Data; D017433:Protein Structure, Secondary; D011509:Proteoglycans / Q000737:chemistry* / Q000302:isolation & purification*; D011994:Recombinant Proteins / Q000737:chemistry; D012542:Scattering, Radiation; D017386:Sequence Homology, Amino Acid; D013696:Temperature; D014461:Ultracentrifugation; D014822:Vitreous Body / Q000378:metabolism
D016326:Extracellular Matrix Proteins; D008024:Ligands; C404108:OPTC protein, human; D011509:Proteoglycans; D011994:Recombinant Proteins; D003094:Collagen; D019791:Guanidine