Comparative Analysis of the Major Polypeptides from Liver Gap Junctions and Lens Fiber Junctions

Gap junctions from rat liver and fiber junctions from bovine lens have similar septilaminar profiles when examined by thin-section electron microscopy and differ only slightly with respect to the packing of intramembrane particles in freeze-fracture images. These similarities have often led to lens fiber junctions being referred to as gap junctions. Junctions from both sources were isolated as enriched subcellular fractions and their major polypeptide components compared biochemically and immunochemically. The major liver gap junction polypeptide has an apparent molecular weight of 27,000, while a 25,000-dalton polypeptide is the major component of lens fiber junctions. The two polypeptides are not homologous when compared by partial peptide mapping in SDS. In addition, there is not detectable antigenic similarity between the two polypeptides by immunochemical criteria using antibodies to the 25,000-dalton lens fiber junction polypeptide. Thus, in spite of the ultrastructural similarities, the gap junction and the lens fiber junction are comprised of distinctly different polypeptides, suggesting that the lens fiber junction contains a unique gene product and potentially different physiological properties

The Influence of extracellular matrix on lens epithelial cell viability

Posterior capsular opacification is the main complication of cataract surgery and results from the proliferation, migration and differentiation of lens epithelial cells remaining in the capsular bag. To better understand this pathological cell behaviour, 1 investigated the interactions between lens epithelial cells and the bovine lens capsule in vitro and their effect on cell viability. As determined by a colorimetric cell proliferation assay, in vitro culture of cells directly on the bovine lens capsule resulted in maintained cell viability in the presence of staurosporine in both lens epithelial cell lines tested, but in neither of the two non-lens cell lines tested. As determined by immunoblotting and reverse-transcriptase polymerase chain reaction (RT-PCR), cell viability on the bovine lens capsule could further be correlated to the presence of both ɑA-crystallin and αB-crystallin expression. A positive correlation of cell viability on the lens capsule with vimentin and HSP27 expression was also found in a smaller set of cell lines. As determined by gelatin zymography and immunoblotting, MMP-2 was expressed by lens epithelial cells, led to the release of FGF-2 and IGF-1 from the lens capsule and correlated with lens epithelial cell viability. Taken together, these results suggest that the lens capsule can act as a store of releasable growth factors available to the lens epithelial cells, with effects on their protein expression and cell viability

Comparing the content of lipids derived from the eye lenses of various species

The lipid content in the eye lens was analyzed and compared among various species in this study. The eye lens lipids of the following species were investigated: cow, horse, duck, and freshwater trout. Additionally, the lipids derived from cataractous bovine lens and from cataractous human eye lens lipoprotein complexes were analyzed. The following lipid classes were detected in clear lenses: cholesterol, sphingomyelin, phosphatidylcholine, phosphatidyletanolamine, and phosphatidylserine. In cataractous bovine lens and in lipoprotein complexes from human nuclear cataract, phosphatidyloinositol and phosphatidyloglycerol were detected. Cholesterol and sphingomyelin, essential for hypothetical formation of cholesterol-rich domains, were the most abundant lipids in the lenses of all investigated species. These two components of eye lens lipid fraction were analyzed quantitatively using thin layer chromatography and spectrophotometric assay; the other lipids were identified qualitatively using thin layer chromatography. (Folia Histochemica et Cytobiologica 2011; Vol. 49, No. 3, pp. 425–430

Proteomic analysis of the bovine and human ciliary zonule

PURPOSE: The zonule of Zinn (ciliary zonule) is a system of fibers that centers the crystalline lens on the optical axis of the eye. Mutations in zonule components underlie syndromic conditions associated with a broad range of ocular pathologies, including microspherophakia and ectopia lentis. Here, we used HPLC–mass spectrometry to determine the molecular composition of the zonule. METHODS: Tryptic digests of human and bovine zonular samples were analyzed by HPLC–mass spectrometry. The distribution of selected components was confirmed by immunofluorescence confocal microscopy. In bovine samples, the composition of the equatorial zonule was compared to that of the hyaloid zonule and vitreous humor. RESULTS: The 52 proteins common to the zonules of both species accounted for >95% of the zonular protein. Glycoproteins constituted the main structural components, with two proteins, FBN1 and LTBP2, constituting 70%–80% of the protein. Other abundant components were MFAP2, EMILIN-1, and ADAMTSL-6. Lysyl oxidase-like 1, a crosslinking enzyme implicated in collagen and elastin biogenesis, was detected at significant levels. The equatorial and hyaloid zonular samples were compositionally similar to each other, although the hyaloid sample was relatively enriched in the proteoglycan opticin and the fibrillar collagens COL2A1, COL11A1, COL5A2, and COL5A3. CONCLUSIONS: The zonular proteome was surprisingly complex. In addition to structural components, it contained signaling proteins, protease inhibitors, and crosslinking enzymes. The equatorial and hyaloid zonules were similar in composition, but the latter may form part of a composite structure, the hyaloid membrane, that stabilizes the vitreous face

ウシ水晶体におけるProtein Carboxyl-o-Methyltransferaseに関する研究

It has been established that the long-lived proteins of the mammalian eye lens slowly accumulate D-aspartyl(D-Asp) and L-isoaspartyl (L-Isoasp) residues as an apparent consequence of aging. Protein carboxyl-o-methyltransferase (PCMT) [EC.2.1.1.24], which is widely distributed in mammalian tissues, has been found to selectively methylate the β-carboxyl group of the uncommon D-Asp and L-Isoasp residues in proteins. Considering these findings, it has been proposed that PCMT plays a role in the repair or degradation of the abnormal amino acid residues that accumulate in aged proteins. PCMT has been detected in the bovine eye lens, however, the enzyme in the lens has not been characterized in detail. In the present study, we studied the properties and structure of the enzyme in the bovine lens. 1. Type II of PCMT (PCMT-II) was purified more than 13,000-fold from the cytosol of bovine lens. The apparent molecular weight of the PCMT-II on SDS-PAGE was approximately 27,000. The activity of type I of PCMT, which was not absorbed on DEAE-cellulose, was extremely low in the bovine lens. 2. The PCMT-II from the lens was composed of at least two molecular species, II a and II b, which were separated by a Mono-Q column. The pI was 5.8 for IIa and 5.6 for IIb. The two molecular species were found to have similar Km and Vmax values. The optimum pH of IIa activity ?was about 6.5, IIb activity showed a clearly lower pH optimum of less than 6.0. 3. The relative rates between PCMT-IIa and IIb activities were very similar with five proteins as methyl accepting substrates. Crystallin, the main protein constituent in the lens, gave the largest methylating rate of any protein examined here. 4. Antibody raised against PCMT-II from bovine lens crossreacted with PCMT-I and II from bovine brain, and PCMT from human erythrocyte. 5. The amino acid compositions of PCMT-IIa and IIb from bovine lens and PCMT-II from bovine brain were very similar. The HPLC profiles of lysylendopeptidase digests of PCMT-IIa and IIb from bovine lens and PCMT-II from bovine brain were also very similar, suggesting that similar seqences are present among these three enzymes. 6. The amino acid sequence in each peak of the lysylendopeptidase digests of PCMT-II a from bovine lens was almost identical with that of PCMT-I from bovine brain and PCMT-II from human and bovine erythrocytes reported by other investigators. These results suggest that PCMT-I and II of various organs from different mammalian species may have nearly identical structures. Therefore, differences in pI and pH optimum between PCMT-IIa and IIb from bovine lens may be due to the structural modification of the enzyme

Study of congenital Morgagnian cataracts in Holstein calves

Cataracts are focal to diffuse opacities of the eye lens causing impaired vision or complete blindness. For bilateral congenital cataracts in Red Holsteins a perfectly cosegregating mutation within the CPAMD8 gene (CPAMD8:g.5995966C>T) has been reported. We genotyped the CPAMD8:g.5995966C>T variant in Holstein calves affected by congenital bilateral congenital cataracts, their unaffected relatives and randomly selected herd mates. Ophthalmological examinations were performed in all affected individuals to confirm a congenital cataract. Whole genome sequencing was employed to screen variants in candidate genes for the Morgagnian cataract phenotype. In the present study, 3/35 cases were confirmed as homozygous mutated and 6/14 obligate carriers. Further 7/46 unaffected animals related with these cases were heterozygous mutated for the CPAMD8:g.5995966C>T variant. However 32 cases with a congenital cataract showed the wild type for the CPAMD8 variant. We did not identify variants in the candidate genes CPAMD8 and NID1 or in their close neighborhood as strongly associated with the congenital cataract phenotype in Holstein calves with the CPAMD8 wild type. In conclusion, the CPAMD8:g.5995966C>T variant is insufficient to explain the majority of Morgagnian congenital cataract phenotypes in Holsteins. It is very likely that congenital bilateral cataracts may be genetically heterogeneous and not yet known variants in genes other than CPAMD8 and NID1 are involved

The structural organization and protein composition of lens fiber junctions.

The structural organization and protein composition of lens fiber junctions isolated from adult bovine and calf lenses were studied using combined electron microscopy, immunolocalization with monoclonal and polyclonal anti-MIP and anti-MP70 (two putative gap junction-forming proteins), and freeze-fracture and label-fracture methods. The major intrinsic protein of lens plasma membranes (MIP) was localized in single membranes and in an extensive network of junctions having flat and undulating surface topologies. In wavy junctions, polyclonal and monoclonal anti-MIPs labeled only the cytoplasmic surface of the convex membrane of the junction. Label-fracture experiments demonstrated that the convex membrane contained MIP arranged in tetragonal arrays 6-7 nm in unit cell dimension. The apposing concave membrane of the junction displayed fracture faces without intramembrane particles or pits. Therefore, wavy junctions are asymmetric structures composed of MIP crystals abutted against particle-free membranes. In thin junctions, anti-MIP labeled the cytoplasmic surfaces of both apposing membranes with varying degrees of asymmetry. In thin junctions, MIP was found organized in both small clusters and single membranes. These small clusters also abut against particle-free apposing membranes, probably in a staggered or checkerboard pattern. Thus, the structure of thin and wavy junctions differed only in the extent of crystallization of MIP, a property that can explain why this protein can produce two different antibody-labeling patterns. A conclusion of this study is that wavy and thin junctions do not contain coaxially aligned channels, and, in these junctions, MIP is unlikely to form gap junction-like channels. We suggest MIP may behave as an intercellular adhesion protein which can also act as a volume-regulating channel to collapse the lens extracellular space. Junctions constructed of MP70 have a wider overall thickness (18-20 nm) and are abundant in the cortical regions of the lens. A monoclonal antibody raised against this protein labeled these thicker junctions on the cytoplasmic surfaces of both apposing membranes. Thick junctions also contained isolated clusters of MIP inside the plaques of MP70. The role of thick junctions in lens physiology remains to be determined

A heterologous expression system for bovine lens transmembrane main intrinsic protein (MIP) in Nicotiana tabacum plants

We have developed a heterologous expression system for transmembrane lens main intrinsic protein (MIP) in Nicotiana tabacum plant tissue. A native bovine MIP26 amplicon was subcloned into an expression cassette under the control of a constitutive Cauliflower Mosaic Virus promoter, also containing a neomycin phosphotransferase operon. This cassette was transformed into Agrobacterium tumefaciens by triparental mating and used to infect plant tissue grown in culture. Recombinant plants were selected by their ability to grow and root on kanamycin-containing media. The presence of MIP in the plant tissues was confirmed by PCR, RT-PCR and immunohistochemistry. A number of benefits of this system for the study of MIP will be discussed, and also its application as a tool for the study of heterologously expressed proteins in general