Characterization of human lens major intrinsic protein structure.
OBJECTIVE
To determine the primary covalent structure of human lens major intrinsic protein (MIP) in lenses of varying age.


METHODS
MIP was isolated from single human lenses of various ages (7- 86 years) by homogenization of the lenses, followed by centrifugation and urea washes of the membranes. Proteins present in the membrane preparation were reduced, alkylated, and cleaved by CNBr. Peptide fragments were fractionated by reverse-phase high-performance liquid chromatography, and the primary structures of the peptides were determined by tandem mass spectrometry and Edman sequencing.


RESULTS
Complete coverage of the human MIP sequence was observed in the form of CNBr fragments. In addition, peptide structures resulting from in vivo heterogeneous N- and C-terminal cleavage were characterized. The amount of intact MIP decreased with lens age; however, the pattern of truncation did not change from 7 to 86 years. The major site of phosphorylation was identified as serine 235. Asparagine residues 246 and 259 were completely deamidated by age 7 years.


CONCLUSIONS
The major structural modifications of human lens MIP have been determined. Human MIP is heterogeneously modified in lenses ranging in age from 7 to 86 years of age by N- and C-terminal truncation, phosphorylation, and deamidation, resulting in decreased levels of native intact MIP with age.
Schey
K L
KL
Department of Cell and Molecular Pharmacology, Medical University of South Carolina, Charleston 29425, USA. scheykl@musc.edu
Little
M
M
Fowler
J G
JG
Crouch
R K
RK
D000293:Adolescent; D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D000375:Aging / Q000502:physiology; D000595:Amino Acid Sequence; D020346:Aquaporins; D002648:Child; D002851:Chromatography, High Pressure Liquid; D003641:Deamination; D005136:Eye Proteins / Q000032:analysis* / Q000302:isolation & purification / Q000378:metabolism; D006801:Humans; D007908:Lens, Crystalline / Q000737:chemistry* / Q000378:metabolism; D008562:Membrane Glycoproteins; D008875:Middle Aged; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000032:analysis; D010766:Phosphorylation; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D020346:Aquaporins; D005136:Eye Proteins; D008562:Membrane Glycoproteins; D010446:Peptide Fragments; C030328:aquaporin 0