Identification of origin of two polypeptides of 4 and 5 kD isolated from human lenses.
OBJECTIVE
To purify crystallin fragments (degraded polypeptides molecular weight < 18 kD) and identify their parent crystallins.


METHODS
The purification of polypeptides with apparent molecular weights of 4 and 5 kD was carried out using three sequential steps: Sephadex G-50 chromatography under denaturing conditions, preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and high-performance liquid chromatography using a C-18 column. The parent crystallins of the two polypeptides were identified by the Western blotting method using polyclonal antibodies raised against individual 4 and 5 kD polypeptides and by comparing N-terminal amino acid sequences of the polypeptides with crystallins.


RESULTS
Two polypeptides of 4 and 5 kD were purified by the three sequential steps as described from water-soluble proteins of lenses from 60-80-year-old donors. Both purified polypeptides showed a single major peak during high-performance liquid chromatography on a C-18 column and also a single band during sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The Western blot analyses showed maximum immunoreactivity of the anti-4 kD polypeptide antibody to a 22 kD species of beta-crystallin, whereas the anti-5 kD polypeptide antibody showed maximum reactivity to only the alpha B crystallin. These results were further confirmed during comparison of the N-terminal amino acid sequences of the two polypeptides with crystallins. Such comparison showed that the 4 kD polypeptide originated from beta A 3/A1 crystallin after cleavage at His187-His188 bond. Further, the 5 kD polypeptide was a fragment of alpha B crystallin that originated after cleavage at Val145-Asn146 bond.


CONCLUSIONS
These results showed that specific bonds of beta A3/A1 and alpha B crystallins are posttranslationally cleaved in vivo to produce 4 kD and 5 kD polypeptides, respectively.
Srivastava
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Missouri Lions Eye Research Foundation, Columbia.
Srivastava
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K
Silney
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D000368:Aged; D000369:Aged, 80 and over; D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D003459:Crystallins / Q000302:isolation & purification* / Q000378:metabolism; D003911:Dextrans; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007908:Lens, Crystalline / Q000737:chemistry* / Q000378:metabolism; D008875:Middle Aged; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000302:isolation & purification / Q000378:metabolism; D010455:Peptides / Q000302:isolation & purification* / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D011817:Rabbits
D003459:Crystallins; D003911:Dextrans; D010446:Peptide Fragments; D010455:Peptides; C025614:sephadex