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Characterization of human lens major intrinsic protein structure
K L Schey1, M Little, J G Fowler
1Department of Cell and Molecular Pharmacology, Medical University of South Carolina, Charleston 29425, USA. scheykl@musc.edu
Purpose:
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.
Insights
Human lens major intrinsic protein (MIP) undergoes age-related structural changes, including truncation, phosphorylation, and deamidation. Intact MIP levels decrease with age, but modification patterns remain consistent across a wide age range.
Area of Science:
- Ophthalmology
- Biochemistry
- Proteomics
Background:
- The human lens major intrinsic protein (MIP) is crucial for lens transparency and integrity.
- Understanding age-related modifications of MIP is essential for comprehending lens aging and cataract formation.
Purpose of the Study:
- To elucidate the primary covalent structure of human lens MIP in relation to aging.
- To identify specific modifications and their prevalence in lenses of different ages.
Main Methods:
- MIP was isolated from human lenses aged 7 to 86 years.
- Proteins were subjected to reduction, alkylation, and cyanogen bromide (CNBr) cleavage.
- Peptide fragments were analyzed using high-performance liquid chromatography (HPLC), tandem mass spectrometry, and Edman sequencing.
Main Results:
- Complete sequence coverage of human MIP was achieved through CNBr fragments.
- Heterogeneous N- and C-terminal cleavage, phosphorylation at serine 235, and deamidation of asparagine residues 246 and 259 were identified.
- Intact MIP levels decreased with age, while truncation patterns remained consistent from 7 to 86 years.
Conclusions:
- The study determined major structural modifications of human lens MIP.
- Human MIP exhibits age-dependent modifications including truncation, phosphorylation, and deamidation.
- These modifications lead to a reduction in native intact MIP levels with increasing age.