Immunodetection of membrane skeletal protein 4.2 in bovine and chicken eye lenses and erythrocytes.
OBJECTIVE
Protein 4.2 is a major erythrocyte membrane skeletal protein, playing an important role in maintaining the integrity and stability of the membrane. It is a transglutaminase-like molecule with no enzymatic cross-linking activity. Several protein 4.2-associated proteins (i.e. band 3, ankyrin, and protein 4.1) and transglutaminase activities have been detected in the lens. The purpose of this study is to find out if protein 4.2 is also expressed in lens fiber membranes.


METHODS
Western blot analysis of cell membranes isolated from bovine and chicken lens fibers and erythrocytes, and immunocytochemistry of frozen sections of bovine and chicken lens fibers were carried out using two protein 4.2-specific antibodies. These two peptide antibodies have been used to identify two alternatively spliced protein 4.2 isoforms in human erythrocyte membranes: the short (P4.2S, or hP4.2(691)) and the long (P4.2L, or hP4.2(721)) isoforms.


RESULTS
Western blot analysis using anti-P4.2(L) antibody demonstrated specific immunoreactive polypeptides in bovine and chicken lens fiber membranes and erythrocyte membranes, co-migrating with hP4.2(721). Immunofluorescence staining of bovine and chicken lenses, using anti-P4.2(L) antibody, revealed specific signals along the cell membranes of cortical fibers. The signals exhibited a unique, patchy pattern along the cortical fiber cell membranes in both cross-sectional and longitudinal views. In cross sections, the labeling of anti-P4.2(L) along the entire cell membranes gave an appearance of a hexagonal shape of fiber cells.


CONCLUSIONS
Protein 4.2, or its analogs, is present in the lens fiber membranes. Its specific staining pattern in the lens fibers suggests that it participates in the architecture of the lens fiber cell membranes, and may play a role in the lens mechanics and pathology.
Sung
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Department of Bioengineering, University of California, San Diego, La Jolla 92093-0412, USA. amysung@bioeng.ucsd.edu
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D000595:Amino Acid Sequence; D000818:Animals; D001798:Blood Proteins / Q000378:metabolism*; D015153:Blotting, Western; D002417:Cattle; D002645:Chickens; D003598:Cytoskeletal Proteins; D004910:Erythrocyte Membrane / Q000378:metabolism*; D005136:Eye Proteins / Q000378:metabolism*; D019084:Fluorescent Antibody Technique, Indirect; D007908:Lens, Crystalline / Q000166:cytology / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism*; D008969:Molecular Sequence Data; D010446:Peptide Fragments
D001798:Blood Proteins; D003598:Cytoskeletal Proteins; D005136:Eye Proteins; D008565:Membrane Proteins; D010446:Peptide Fragments; C044791:erythrocyte membrane band 4.2 protein