Malkhan Katar

Heat shock proteins of adult and embryonic human ocular lenses

We investigated the presence and distribution of heat shock proteins, HSP‐70 [Horwitz, J. 1992. Proc Natl Acad Sci 89:10449–10453], HSP‐40, HSc‐70, HSP‐27, and αβ‐crystallin in different regions of adult and fetal human lenses and in aging human lens epithelial cells. This study was undertaken because heat shock proteins may play an important role in the maintenance of the supramolecular organization of the lens proteins. Human adult and fetal lenses were dissected to separate the epithelium, superficial cortex, intermediate cortex, and nucleus. The water soluble and insoluble protein fractions were separated by SDS–PAGE, and transferred to nitrocellulose paper. Specific antibodies were used to identify the presence of heat shock proteins in distinct regions of the lens. HSP‐70 [Horwitz, 1992 ], HSP‐40, and HSc‐70 immunoreactivity was mainly detected in the epithelium and superficial cortical fiber cells of the adult human lens. The small heat shock proteins, HSP‐27 and αβ‐crystallin were found in all regions of the lens. Fetal human lenses showed immunoreactivity to all heat shock proteins. An aging study revealed a decrease in heat shock protein levels, except for HSP‐27. The presence of HSP‐70 [Horwitz, 1992 ], HSP‐40, and HSc‐70 in the epithelium and superficial cortical fiber cells imply a regional cell specific function, whereas the decrease of heat shock protein with age could be responsible for the loss of optimal protein organization, and the eventual appearance of age‐related cataract. J. Cell. Biochem. 84: 278–284, 2002.

Lipid composition of chick lens fiber cell gap junctions

Chick lens fiber cell gap junctions were isolated to homogeneity by the urea-deoxycholate method, characterized ultrastructurally and biochemically, and their lipid composition determined by quantitative thin layer chromatography (TLC). The junctions were estimated to comprise about 52% of the lens fiber plasma membrane. Unlike the junctions of other organs, the lens gap junctions were found to contain sphingomyelin. The cholesterol/phospholipid molar ratio was 2.1 for total fiber membranes but 3.1 for the fiber gap junctions. The levels of major phospholipids in decreasing order were SPH, PC, PE, PI for fiber junctions and PE, SPH, PC, PI for total fiber membranes. The gap junctions were found to contain about 57% of the total fiber cholesterol and 53% of the total fiber sphingomyelin. The high cholesterol and sphingomyelin content suggests that lens fiber gap junctions constitute highly rigid membrane regions conferring significant constraints to the movement of their intramembrane particles (connexons) in the plane of the membrane. The findings help to explain the resistance to the crystallization of their connexons, observed so far only in lens gap junctions.

Limited proteolysis of MP26 in lens fiber plasma membranes of the U18666A-induced cataract in rats.

Most of the animals treated with U18666A every other day beginning at one-day of age developed permanent nuclear cataracts by 3-4 weeks of age. Lens fiber plasma membranes were isolated from cortical and nuclear areas of untreated controls, treated but clear, and treated cataractous lenses, and analyzed by SDS-PAGE. MP26 was the major intrinsic polypeptide in the plasma membranes of both cortical and nuclear fibers of control lenses. MP26 was largely replaced by MP23-24 in the plasma membranes of nuclear fibers of treated but clear lenses, and in the membranes of both cortical and nuclear fibers of cataractous lenses.

Heat shock proteins of chicken lens

The presence of heat shock proteins HSP‐40, HSP‐70, and HSc‐70 in adult and embryonic chicken lenses were determined. The epithelium, cortex, and nucleus of adult chicken lens were separated and tested for the presence of heat shock proteins (hsps) by western blot, using specific antibodies for HSP‐40, HSP‐70, and HSc‐70. Water soluble (WSF) and water insoluble fractions (WIF) of embryonic chicken lenses were isolated and tested for the presence of HSP‐40, HSP‐70, and HSc‐70 by immunoblot. Embryonic chicken lens sections were also analyzed for the presence of heat shock proteins by immunoflorescence technique. Data obtained from these experiments revealed that HSP‐40, HSP‐70, and HSc‐70 are present in all areas of both adult and embryonic chicken lens. Presence of hsps protein in the deep cortex and nucleus is intriguing as no detectable metabolic activities are reported in this area. However it can be proposed that hsps HSP‐40, HSP‐70, and HSc‐70 can interact with protein of these areas and protect them from stress induced denaturation. J. Cell. Biochem. 82:409–414, 2001.

Human beta crystallins: regional and age related changes

The composition of human beta-crystallins displayed specific changes with age and region of the lens. 27 kD and 29 kD human beta-crystallin subunits were singled out for study. The 29 kD beta-crystallin subunit constituted approximately 10% of the total lens crystallins at 8 months of fetal life. Its accumulation decreased steadily to 3.3% during postnatal year 1, to 0.5% by year 5 and to 0.3% thereafter. At all postnatal ages, however, it persisted mainly in the superficial fibers. Thus in a 17-years old lens it made up 1.3% of the superficial fiber soluble protein but was already absent from deep cortical and nuclear fibers. The 27 kD subunit increased steadily from 3.5% at 8 months fetal to 7% at year 5; it then decreased steadily to 1.2% in the 86-year old lens. It persisted in all regions of the lens but decreased markedly in the deep cortical and nuclear fibers with increasing age beginning at 5-17 years of age. Studies on the oligomeric structure of human beta-crystallin must take into account age-related changing quantitative patterns in the subunit polypeptide composition of this lens protein.

NCAM of the mammalian lens.

NCAM is present in the plasma membranes of human and rat lens epithelial cells and superficial fiber cells. The predominant isoform in epithelial cells is NCAM 140, while NCAM 120 appears only in the superficial fiber cells. The immunofluorescence patterns are consistent with a decreasing concentration of NCAM associated with fiber cell differentiation.

ERM proteins of the lens.

Ezrin and radixin and protein 4.1 were detected in the lens of the eye. These proteins were mainly present in the young elongating cortical fiber cells and localized to the plasma membranes. Moesin was not detected. Ezrin, radixin, and protein 4.1 provide another means whereby actin is linked to the plasma membrane in addition to the known adherens junctions in the lens.

Associated proteins of lens adherens junction

Cytoplasmic proteins associated with adherens junctions were identified in the chicken ocular lens. The catenins, α, β, and γ, were present in epithelial and fiber cells, although their pattern of distribution changed with fiber cell differentiation. The sharp decline in α‐catenin with fiber cell formation and the increasing Triton‐insolubility of N‐cadherin suggests that another subtype of α‐catenin exists in the lens. J. Cell. Biochem. 86: 700–703, 2002.

A heat shock transcription factor like protein in the nuclear matrix compartment of the tissue cultured mammalian lens epithelial cell.

This investigation characterizes a prominent nuclear matrix protein isolated from tissue cultured mouse lens epithelial cells. The nuclear matrix protein was isolated using a modified Penman technique. Total nuclear matrix proteins were further separated by SDS‐polyacrylamide gel electrophoresis. The SDS‐PAGE profile of the nuclear matrix proteins displayed a prominent doublet band at 60 kDa region. Nonequilibrium 2D gel electrophoresis revealed that this protein is a basic nuclear protein. This 60 kDa protein was further characterized by comparing its internal peptide amino acid sequence with known protein sequence using the BLAST technique, and this study demonstrated that 60 kDa nuclear matrix protein displays significant sequence similarity with Xenopus Laevis heat shock transcription factor. We also raised antibodies against 60 kDa nuclear matrix protein. Immunofluorescence, studies showed that this 60 kDa nuclear matrix protein preferably decorates nucleus, and puncted pattern of fluorescence suggest presence of this protein in the discrete areas of the nucleus. Heat shock transcription factors upregulate synthesis of heat shock proteins and many of these protein act as molecular chaperones. Thus, presence of a nuclear matrix protein with significant sequence similarity with heat shock transcription factor suggests sustained heat shock protein synthesis in the mouse lens cells. J. Cell. Biochem. 80:382–387, 2001.

Paralemnin of the lens

Paralemmin was identified in the chicken lens as a protein with mol. wt 65 kDa and a splice variant of 60 kDa, both soluble in Triton X‐100. Paralemmin is localized to the plasma membrane of fiber cells, and was not detected in the annular pad cells. Thus in the chick lens it is another feature of fiber cell differentiation. Its localization to the short side of the fiber cell and the sites of fiber cell interlocking suggests that paralemmin may play a role in the development of such interdigitating processes. J. Cell. Biochem. 89: 917–921, 2003.