Identification of type VI collagen in the trabecular meshwork and expression of its mRNA by trabecular cells.
To investigate the nature of the 140 kDa glycoprotein in the trabecular meshwork, polypeptides were extracted with either urea/sodium dodecyl sulfate (SDS)/beta-2-mercaptoethanol (BME) or guanidine hydrochloride followed by pepsin digestion. After electrophoresis and immunoblotting with anti-type-VI-collagen antibodies, a single fraction of molecular weight 140 kDa was identified in the urea/SDS/BME extracts. Pepsin solubilization revealed two immunoreactive fractions (molecular weights 75 and 85 kDa) that comigrated with purified, pepsin-solubilized type VI collagen. By using the polymerase chain reaction (PCR) and primers specific for the alpha 2(VI) chain of type VI collagen, a single PCR product was obtained, which corresponded to the expected size of 137 base pairs, from the total RNA extracted from the trabecular meshwork ex vivo. Southern hybridization with the antisense oligonucleotide probe of the alpha 2(VI) chain confirmed that the amplified sequence was specific. The results show that the trabecular meshwork contains a significant amount of type VI collagen and that trabecular cells express the mRNA coding for the alpha 2(VI) chain of this glycoprotein. The presence of type VI collagen in the trabecular meshwork is implicated in cell-extracellular matrix interactions at this site, and its abnormal accumulation in glaucomatous and aging eyes probably signifies a defect in the function of the trabecular cells in these eyes.
Tripathi
B J
BJ
University of South Carolina School of Medicine, Columbia 29208.
Hansen
M
M
Li
J
J
Tripathi
R C
RC
D000595:Amino Acid Sequence; D000818:Animals; D015139:Blotting, Southern; D003094:Collagen / Q000032:analysis*; D004591:Electrophoresis, Polyacrylamide Gel; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000032:analysis*; D013552:Swine; D014129:Trabecular Meshwork / Q000737:chemistry*
D012333:RNA, Messenger; D003094:Collagen