Characterization of human β-crystallin subunits in newborn human lens by cDNA sequence and mass spectrometric analysis

K. J. Larapi, Larry David, Z. Ma, J. B. Smith

Research output: Contribution to journalArticle

1 Scopus citations

Abstract

Purpose: To determine the previously unknown sequences of human β-crystallins, correct the errors in previously reported sequences, confirm these sequences by mass spectrometric analysis, and characterize truncated forms of β-crystallins present at birth. Methods: Crystallins were isolated from donors of less than one week of age and identified by a combination of 2-dimensional electrophoresis and Edman sequencing. Either partial or full length cDNAs of the newly identified crystallins were sequenced and the masses of their deduced amino acid sequences were confirmed by mass spectrometry. Crystallins were prepared for mass spectrometry by either gel filtration and reversed phase chromatography or by electroelution from 2-dimensional gels. Results: The positions of all major human crystallins were determined on 2dimensional gels. Newly identified crystallins included: βAl, βA3, βA4, and βB3. The full length cDNA of βA4, 5' region of βB3 cDNA, and corrected sequence of βA3/Al cDNA were determined. The resulting deduced amino acid sequences were then confirmed by mass spectrometry. A partially truncated form of βA3, missing 22 residues from its N-terminus was also detected by Edman sequencing and then confirmed by mass spectrometry. Conclusions: Conformation of the presence and correct primary structure of all human β-crystallins will allow examination of the age related changes in these proteins. βA3/Al, as well as βBl are the most proteolytically labile crystallin subunits in the human lens. Modified forms of both proteins are already present at birth. Cleavage of both proteins between asparagine and proline residues in their N-terminal extensions suggested that a single proteolytic activity may be responsible.

Original languageEnglish (US)
JournalInvestigative Ophthalmology and Visual Science
Volume38
Issue number4
Publication statusPublished - 1997

    Fingerprint

ASJC Scopus subject areas

  • Ophthalmology

Cite this