Amino acid sequence analysis of proteins in the human corneal stromal cell membrane.
Plasma membrane proteins from human corneal stromal fibroblasts were isolated and separated by two-dimensional polyacrylamide gel electrophoresis. Separated polypeptides were electroeluted onto polyvinylidene difluoride (PVDF) membranes and individual polypeptides were subjected to NH2-terminal amino acid sequence analysis. Of a total of 33 polypeptides sequenced, 26 were found to be blocked at their NH2-terminus. Seven major membrane polypeptides were sequenced and further analyzed. One polypeptide, designated #18, was determined to be homologous to the beta subunit of prolyl hydroxylase/protein disulfide isomerase/thyroid hormone-binding protein. The other six polypeptides were found to have no significant sequence homology with any known polypeptides, as revealed by a protein data base homology search. Polypeptide Bands #90, #102, and #103 were found to have the same NH2-terminal amino acid sequence and the same overall molecular weight, yet separated from one another according to pI. These three polypeptides probably arose from differential posttranslational modification of the same original protein. Synthetic peptides were prepared from the #18 and #19 sequence and antibodies were produced. Immunostaining of cultured human corneal stromal cells and frozen sections of corneas demonstrated that these membrane polypeptides were present in corneal keratocytes, both in vitro and in vivo. Antibody against #18 stained fixed cultured corneal fibroblasts in a very fibrous pattern, with more intense staining in the perinuclear region of the cell, while antibody against #19 stained the cell surface in a much more uniform pattern. In sections of human cornea, both antibodies stained only the keratocytes in the stroma, but they also appeared to stain epithelial cells.
Ahmad
M
M
Emory University School of Medicine, Department of Ophthalmology, Atlanta, GA 30322.
Church
R L
RL
D000595:Amino Acid Sequence; D002462:Cell Membrane / Q000378:metabolism; D003319:Corneal Stroma / Q000378:metabolism*; D004797:Enzyme-Linked Immunosorbent Assay; D005136:Eye Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D006801:Humans; D008969:Molecular Sequence Data; D013194:Staining and Labeling
D005136:Eye Proteins