Identification and characterization of structural components in the mammalian vitreous

Vitreous humor is a unique extracellular matrix which is primarily composed of collagen fibrils and hyaluronan molecules. These two major structural components form independent networks and the two networks potentially interact with each other to give the vitreous its unique transparent gel-like property. Altered interactions between the two networks may disrupt the overall organization of the vitreous matrix and may result in the aggregation of the collagen fibrils and collapse of the vitreous gel. This often results in posterior vitreous detachment which potentially can lead to retinal detachment if the vitreous remains locally attached to the retina.;A number of noncollagenous proteins and glycoproteins may contribute to the molecular organization of the vitreous and sustain its unique gel-like property. In order to understand the macromolecular organization of mammalian vitreous, I have investigated its structural components and have identified a novel protein called vitrin. This protein is released from a pellet of collagen fibrils obtained from bovine vitreous by a high salt solution. When analyzed on SDS-PAGE, the high salt extract of vitreous collagen fibrils showed a unique band with a molecular weight of 90 kDa. The amino terminus and several internal tryptic peptides of vitrin all demonstrated unique amino acid sequences. A fragment of bovine cDNA to vitrin of length 1.35 kb was generated from the peptide sequences using degenerate oligonucleotides and the reverse transcriptase polymerase chain reaction (RT-PCR). From this bovine sequence, several Expressed Sequence Tags (ESTs) to human vitrin were found in GenBank. The full length cDNA sequence of human vitrin was generated by PCR both by using specific primers designed from these ESTs and by the Rapid Amplification of cDNA Ends (5;We have also identified a second protein called amyloid protein precursor (APP) as a component of the vitreous gel. It contains a Kunitz protease inhibitor (KPI) domain and was shown to have protease inhibitory activity. APP could potentially play a key role in regulating the level and turnover of extracellular matrix components within the vitreous.;Our experimental findings provide us with a new understanding of the organization of the vitreous gel and may lead to new insights into the molecular mechanisms leading to vitreous syneresis or liquifaction, posterior vitreous detachment, and retinal detachment.