Versican/PG-M isoforms in vascular smooth muscle cells.

The expression of increased amounts of proteoglycans in the extracellular matrix may play a role in vascular stenosis and lipid retention. The large chondroitin sulfate proteoglycan versican is synthesized by vascular smooth muscle cells (SMCs), accumulates during human atherosclerosis and restenosis, and has been shown to bind LDLs. We recently demonstrated that adult rat aortic SMCs express several versican mRNAs. Four versican splice variants, V0, V1, V2, and V3, have recently been described, which differ dramatically in length. These variants differ in the extent of modification by glycosaminoglycan chains, and V3 may lack glycosaminoglycan chains. In this study, we characterized versican RNAs from rat SMCs by cloning, sequencing, and hybridization with domain-specific probes. DNA sequence was obtained for the V3 isoform, and for a truncated V0 isoform. By hybridization of polyadenylated RNA with domain-specific probes, we determined that the V0, V1, and V3 isoforms are present in vascular SMCs. We confirmed the presence of the V3 isoform in polyadenylated RNA and in RT-PCR products by hybridization with an oligonucleotide that spans the splice junction between the hyaluronan-binding domain and the epidermal growth factor-like domain. In addition, a novel splice variant was cloned by PCR amplification from both rat and human SMC RNA. This appears to be an incompletely spliced variant, retaining the final intron. PCR analysis shows that this intron can be retained in both V1 and V3 isoforms. The predicted translation product of this variant would have a different carboxy-terminus than previously described versican isoforms.

Ozone alters the expression of tenascin-C in cultured primate nasal epithelial cells.

Tenascin-C is an extracellular matrix component which is transiently expressed in association with epithelial cell detachment, proliferation, and migration. This molecule has been identified in respiratory tissue, but little is known about the cellular source of tenascin-C or the factors that regulate its production. Since air pollutants are known to disrupt epithelial integrity, we investigated the regulation of tenascin-C in response to 0.3 ppm ozone in differentiated primate nasal epithelial cells in culture at an air-medium interface. The expression of tenascin-C was upregulated in response to ozone, as determined by Northern blot analysis, Western blotting, and immunofluorescent staining. In contrast, there was no change in the mRNA levels for versican, biglycan, perlecan, or collagen type I. Reduced cellular attachment to the substrate was evident in ozone-treated cultures in association with tenascin-C deposition at the interfaces between cells and basal surfaces. The presence of tenascin-C on denuded areas of the matrix suggests that tenascin-C may have been instrumental in the loss of patches of cells. The modulation of tenascin-C synthesis and distribution may play a significant role in the response of respiratory epithelial cells to ozone exposure.

Dynamic expression patterns of tenascin, proteoglycans, and cell adhesion molecules during human hair follicle morphogenesis.

The development of skin appendages such as hair, feathers, and teeth is brought about by reciprocal interactions between epidermal and mesenchymal tissues and is thought to be influenced in part by cell adhesion molecules and components of the extracellular matrix. The developmental distributions of tenascin, neural cell adhesion molecule (NCAM), E-cadherin, intercellular adhesion molecule 1 (ICAM-1), chondroitin sulfate proteoglycan (CSPG), and the heparan sulfate proteoglycan perlecan were studied in relation to hair follicle morphogenesis in fetal human skin. Tenascin first appeared in developing skin in focal concentrations at the epidermal-mesenchymal interface, just prior to, and presumably correlated with, hair follicle initiation. Tenascin immunostaining remained prominent in the basement membrane zone and extracellular matrix of the follicle sheath during subsequent morphogenetic stages. Two forms of tenascin (M(r) 250 x 10(3) and 280-300 x 10(3)), were revealed by Western blots of skin extracts. NCAM immunolabeling was initially present throughout the dermis, and became progressively restricted to the dermal condensation and the follicle sheath. Western blot analysis revealed an isoform of NCAM (M(r) 160 x 10(3)) which lacked polysialic acid. At all stages, E-cadherin staining was diminished on follicle cells situated adjacent to the basement membrane, relative to cells in the follicle interior. Follicle-specific immunostaining for ICAM-1 was transient, appearing only at the pre-germ and hair germ stages of development. Antibodies to three distinct CSPG determinants revealed unique immunolabeling patterns following follicle initiation: One CSPG epitope co-distributed with tenascin in the follicle basement membrane and follicle sheath extracellular matrix; one CSPG epitope was similarly expressed, and was also found on follicle epithelial cells; and the third CSPG determinant was noticeably absent from the follicle sheath during elongation of the developing appendage. Perlecan was concentrated in the dermal papilla, in addition to its distribution in all skin basement membranes. A model for how these diverse molecules may interact to influence human hair follicle morphogenesis is presented.

Comparative studies of beta s-crystallins from human, bovine, rat and rabbit lenses.

Soluble extracts from young bovine, human, rat and rabbit lenses were fractionated by high resolution size-exclusion chromatography to demonstrate the existence of three discrete size-classes of monomeric crystallins in each species. These were identified by ion exchange chromatography, amino acid analysis, SDS electrophoresis and isoelectric focusing as the beta s-, gamma A- and gamma B-crystallins. Conventional SDS electrophoretic analysis of these proteins revealed apparent Mr values of about 23kD, 22kD and 19kD, respectively. Similar analysis in the presence of 6 M urea showed the proteins all co-migrated with an apparent Mr of about 20,500, which is far more consistent with the molecular weights calculated from beta s- and gamma-crystallin sequence data. Amino acid compositions of all the beta s samples indicate a high degree of homology to the bovine protein, whose sequence is known. The different species beta s-crystallins showed other general similarities in size, charge, thiol content and secondary structural properties. On the other hand, near UV CD and fluorescence emission and energy transfer measurements indicate that these proteins have subtle yet significant differences in their tertiary structures. Unlike the gamma-crystallins, the secondary structure of all of the beta s samples is completely denatured in the presence of 8 M urea at 20 degrees C.

Oxidative modifications to crystallins induced in calf lenses in vitro by hydrogen peroxide.

Calf lenses which are incubated in solutions of 1-150 mM H2O2 for 24 hr remain clear at 20 degrees C. While insoluble lens protein increases by at most 2-3%, we find extensive oxidation of exposed protein thiols, major shifts in the size distribution of crystallins, and progressive generation of more acidic polypeptides. Some of these oxidative modifications are reversible with reducing agent. beta H-Crystallins are particularly susceptible to oxidation: disulfide-bonded soluble aggregates form at low H2O2 levels, while irreversible dissociation to beta L-crystallins occurs at high H2O2 concentration. The gamma-crystallins are particularly prone to charge modification. In contrast, the size and charge distributions of alpha-crystallins appear to be virtually unaffected by H2O2.

Optimal resolution of eye lens gamma-crystallins by cation-exchange high-performance liquid chromatography on SynChropak CM300.

Cation-exchange high-performance liquid chromatography on SynChropak CM300 in Tris-acetate buffers of pH 5-7, using sodium acetate gradients, produces an excellent separation of the various gamma-crystallin gene products and their post-synthetically modified forms from eye lens. With a single analysis of total lens extract, the gamma-crystallins can be resolved, quantified and collected for amino acid analysis. Experimental conditions are presented for optimal resolution of individual human, rat, bovine and dogfish shark gamma-crystallins. Applications presented include determinations of different synthesis of gamma-crystallins and chemical modification (oxidation by hydrogen peroxide) in situ.

Rat lens gamma-crystallins. Characterization of the six gene products and their spatial and temporal distribution resulting from differential synthesis.

We have isolated, purified and characterized six individual gamma-crystallin polypeptides present in the rat lens. Comparison of their amino acid compositions with the known structure of the six gamma-crystallin genes permits a one-to-one correspondence to be made between each protein synthesized and the encoding gene. This demonstrates that each of the six genes is actually expressed in vivo. Two classes of three gamma-crystallins each, which we have designated classes gamma ABC and gamma DEF, are known to exist, on the basis of internal sequence homology. We have measured the temperature-dependent phase-separation characteristics of solutions of the six purified gamma-crystallins, and find that the three members of the gamma DEF class (gamma 2-2, gamma 3-1 and gamma 4-1) are all cryo-proteins with relatively high phase-separation temperatures, whereas the three gamma ABC crystallins (gamma 1-1, gamma 1-2 and gamma 2-1) do not show phase separation above -7 degrees C. We have measured the spatial distribution in rat lens of each of the alpha-, beta- and gamma-crystallins as a function of age from 1 to 420 days, using size-exclusion and ion-exchange high-pressure liquid chromatography (HPLC). Our findings in the cortical layer permit us to establish the differential synthesis of each of the crystallins during lens development. Particular attention has been devoted to the spatial and temporal distribution of the six individual gamma-crystallins. Up to birth, synthesis of the three components of the gamma DEF class predominates, and in particular that of gamma 2-2. In subsequent development the three components of the gamma ABC class assume a greater proportion of monomeric crystallins synthesized, while beta s-crystallin synthesis predominates in late development. Our analysis of different layers within single lenses provides novel information on spatial gradients of the water-soluble and water-insoluble protein fractions as a function of age. We consider the consequences of these findings for lens transparency and opacity in both rat and mouse lens. We show that the high concentrations of gamma DEF-crystallins appear to be responsible for the opacity known to occur in young rat lenses. We conclude from these observations that close control of the differential synthesis of gamma-crystallins plays an important role in maintaining lens transparency during development.

Heterogeneity of gamma-crystallins from spiny dogfish (Squalus acanthias) eye lens.

Mammalian lenses contain multiple gamma-crystallin gene products, which are differentially synthesized during lens development. We now report the isolation and characterization of multiple gamma-crystallins from lenses of adult spiny dogfish (Squalus acanthias) aged about 20-30 years. About 50% of total lens protein solubilized in 50 mM phosphate, pH 7.0; about 25% of this soluble fraction consists of gamma-crystallins as determined by gel filtration. These gamma-crystallins appear homogeneous with respect to molecular weight (approximately equal to 20,000) on SDS-polyacrylamide gels, but their isoelectric points range from below pH 6 to above 10. Preparative cation-exchange chromatography on SP-Sephadex at pH 4.8 resolves four major subfractions, while anion-exchange on DEAE-cellulose at pH 9.5 resolves seven subfractions. Although these procedures separate basic from acidic polypeptides, most of these gamma-crystallin subfractions still consist of polypeptide mixtures, as determined by ion-exchange HPLC and isoelectric focusing. Analytical cation-exchange HPLC on SynChropak CM300 at pH 6.0 resolves at least 10 different gamma-crystallin components. Amino acid compositions of all the subfractions are similar, yet distinct in the sense that three subclasses can be distinguished. Sulfhydryl residues range from three to six per chain, most of which are buried. The large heterogeneity of gamma-crystallins in adult lens may result from different gene products in combination with post-translational modification.

Human lens gamma-crystallins: isolation, identification, and characterization of the expressed gene products.

We have isolated the individual gamma-crystallins expressed in young human lenses and identified with which of the six known human gamma-crystallin genes they each correspond. We find that at least 90% of the gamma-crystallins synthesized in the young human lens are the products of genes gamma G3 and gamma G4. We demonstrate that gamma G4-crystallin undergoes a temperature-dependent phase separation, and we have measured the low-concentration branch of its coexistence curve (phase separation temperature vs. concentration) up to about 40 mg/ml. By comparison, we found no evidence of gamma G3-crystallin phase separating, even at lower temperatures and higher concentrations. This is consistent with predictions based on sequence homology between human and rat gamma-crystallins. The implications of these findings for human inherited and senile cataracts are considered.

Superior resolution of gamma-crystallins from microdissected eye lens by cation-exchange high-performance liquid chromatography.

A novel procedure is presented for the rapid quantitative analysis of eye lens gamma-crystallins and beta s-crystallin by ion-exchange high-performance liquid chromatography on Synchropak CM300. At least six different gamma-crystallin gene products can be resolved from the soluble fraction of calf lens extract. This method is applicable to the analysis of microsections from individual lenses, and can be used to rapidly characterize spatial variations in gamma-crystallin composition which occur with aging and cataractogenesis.

U1 small nuclear RNA-like sequences in human high molecular weight RNA.

Human U1 small nuclear RNA synthesis was shown earlier to be very sensitive to UV radiation. This led us to test for the possible presence of U1 RNA-like sequences in large RNAs. Human RNA was analyzed in gel blots hybridized with U1 DNA probes. A high molecular weight, heterodisperse RNA population was detected, which hybridizes both to a U1 RNA-coding region single-stranded DNA probe, and to a U1 gene fragment that contains only 6 nucleotides of flanking sequence. These large RNAs can be hybrid selected using immobilized U1 DNA, and have an average size of several kilobases. Additional observations support the claims that the high molecular weight RNA hybridization signal is not an aggregation artifact and that it is sequence specific.