Changes in the components of extracellular matrix and in growth properties of cultured aortic smooth muscle cells upon ascorbate feeding.

Culture conditions can modify the composition of the extracellular matrix of cultured calf aortas smooth muscle cells. In the absence of ascorbate the major components of the matrix are microfibrillar proteins; deposition of collagen occurs upon ascorbate supplementation and, with increased time of exposure of cells to ascorbate, collagen becomes the dominant protein of the extracellular matrix (greater than 80%). Collagen accumulation follows a sigmoidal time-course, suggesting that it is a cooperative phenomenon. Covalent crosslinks are not required for collagen accumulation in the matrix. Microfibrillar proteins and increased amounts of proteoglycans and fibronectin accumulate concurrently with collagen but elastin deposition was not observed either with or without ascorbate feeding. Addition of ascorbate leads to a general stimulation of incorporation of [14C]proline into cellular protein and to changes in cell growth parameters and morphology: cell-doubling time decreases from 62 to 47 h and plating efficiency increases approximately fourfold. We conclude that the composition of the extracellular matrix assembled by cultured cells is subject to experimental manipulation and that changes in endogenously deposited matrix may have significant effects on cellular functions.

Recommendations for locus-specific databases and their curation.

Expert curation and complete collection of mutations in genes that affect human health is essential for proper genetic healthcare and research. Expert curation is given by the curators of gene-specific mutation databases or locus-specific databases (LSDBs). While there are over 700 such databases, they vary in their content, completeness, time available for curation, and the expertise of the curator. Curation and LSDBs have been discussed, written about, and protocols have been provided for over 10 years, but there have been no formal recommendations for the ideal form of these entities. This work initiates a discussion on this topic to assist future efforts in human genetics. Further discussion is welcome.

Preparation of cyanogen bromide fragments of MM, NN, and MN glycoproteins (glycophorins) from human erythrocyte membranes of single donors.

A procedure is described for the preparation of three cyanogen bromide fragments of the MM, NN, or MN glycoprotein (glycophorin) of the human erythrocyte membranes, from erythrocytes of single donors. The fragments are obtained in pure form and excellent yields by employing procedures which include proteolytic inhibitors during membrane processing, thorough delipidation of the glycoprotein, and CNBr cleavage conditions which lead to quantitative fragmentation without loss of carbohydrates. A phenol-urea extraction resolves the two glycopeptide fragments from the carbohydrate-free fragment. The two glycopeptides are further purified by Bio-Gel P-6 and P-100 chromatography. The three fragments include the amino terminal 8 residue glycopeptide, a large glycopeptide form the middle of the molecule which bears the Asn-linked oligosaccharide and 8--9 O-glycosidically linked units, and a carboxyl terminal, carbohydrate-free, approx. 50 residue fragment. Their amino acid and carbohydrate composition, and size, are in close agreement with the sequence data of Tomita, M., Furthmayr, H. and Marchesi, V.T. (Biochemistry (1978), 17, 4756--4770). The fragments represent three well delineated portions of the glycoprotein molecule.

Isolation and characterization of glycoproteins from canine tracheal pouch secretions.

Canine tracheal pouch secretions were solubilized with 1% sodium dodecyl sulfate and visualized by sodium dodecyl sulfate-agarose-acrylamide gel electrophoresis. Intact mucus, and water-soluble and insoluble fractions of mucus were shown to be composed of high molecular weight glycoproteins (Mr greater than or equal to 3 . 10(6)) and three major classes of proteins of lower molecular weight (Mr approximately 4 . 10(5), 2 . 10(5), and 6 . 10(4)). When the mucus secretions were further treated with a reducing agent, the glycoproteins were dissociated into subunits which appeared on the gel as three discrete bands. Separation of the high molecular weight glycoproteins from the other proteins was achieved by gel filtration on Biogel A-15m in the presence of 1% dodecyl sulfate following reduction and alkylation of mucus. These glycoproteins were further resolved, using DEAE cellulose chromatography in the presence of 6 M urea, into two protein fractions. Both fractions contained approximately 87% carbohydrate, high amounts of serine and threonine but differed significantly in contents of N-acetyl glucosamine and sialic acid; their mobility on gel electrophoresis was also different. Significant contents of cysteine were noted in both fractions. Results of this study indicate that the canine tracheal pouch preparations provide normal tracheal secretions which bear similarity in structure to the tracheobronchial secretions obtained from human patients.

Collagen accumulation in heart ventricles as a function of growth and aging.

Increase in resting tension of left ventricular papillary muscle with age has been attributed to the amount of collagen present. We therefore studied the total amount and structure of myocardial collagen as a function of age in the hearts of male Fischer 344 rats. Using amino acid analysis and quantification of hydroxyproline, we showed that collagen accumulates in relation to ventricular protein after 3 months of age and continues in that mode with increased age of the animal, levelling off at 22 months. In this strain of rats, collagen increased in the left ventricle from 5.5% of total protein in a 1 month old animal to approximately 12% in 22 and 26 month old animals; in the right ventricle the increase was from 7% in the 1 month old animal to approximately 19.5% in 22-26 month old animals. The larger percentages of collagen in the right ventricle relative to the left agree with findings of others. Collagen accumulates in intrinsic collagenous structures where the pre-existing fibres are thickened and are more extensive. These structures were detected with light microscopy and scanning electron microscopy and include perimysial weaves, coiled perimysial fibres and struts. Regions of fibrosis were also increased in size and volume in older animals.

Ultrastructure and staining properties of aortic microfibrils (oxytalan).

Microfibrils are the insoluble, 10- to 12-nm components of the extracellular matrix that are involved in elastogenesis. Reports of their ultrastructure vary: they have been described as tubular and beaded and as nontubular filaments that are devoid of any periodicity. Ultrastructurally, microfibrils resemble oxytalan fibers that have been observed in peridontal membranes, skin, and other locations. Whether microfibrils have the staining characteristics of oxytalan is difficult to determine in tissues because available light microscopic stains also stain elastin. Calf aortic smooth muscle cells grown in media without added ascorbate provide a unique model for examining the ultrastructure and staining characteristics of chemically defined microfibrils. Microfibrils are the predominant insoluble extracellular protein in such cultures, which do not deposit collagen or elastin. These studies demonstrate that microfibrils are tubular structures with 10- and 12-nm striations and have the same staining characteristics as oxytalan, reacting with aldehyde fuchsin and orcein after oxidation. Microfibrillar protein is enriched in glutamic and aspartic acids and the electron density of microfibrils is enhanced by fixation in the presence of cationic dyes. In such preparation, microfibrils are made visible within the core of amorphous elastin as well as in regions that are free of elastin. The widespread distribution of microfibrils (oxytalan) indicates that their function extends beyond elastogenesis. Their localization within tissues suggests that they serve as an elastic attachment protein in sites that are subject to mechanical stress.

Extracellular matrix microfibrils are composed of core proteins coated with fibronectin.

Extracellular proteins of cultured calf aortic smooth muscle cells consist predominantly of microfibrils 10-20 nm in diameter typical of "elastin-associated" microfibrils described in many tissues. Chemical and immunochemical evidence is presented that microfibrils consist of at least two proteins: core protein and fibronectin. Insoluble proteins of the microfibrils were obtained in the form of a pellet and antibodies raised in rabbits against these components. The antisera reacted with the insoluble microfibrillar proteins and with soluble fibronectin in enzyme-linked immunosorbent assay, and immunostained the extracellular microfibrils in cultured cells. An immunoglobulin (Ig) fraction was prepared and absorbed with fibronectin. The absorbed IgG retained its reactivity with the microfibrillar proteins but was no longer reactive with soluble fibronectin. Immunofluorescence studies were carried out using the absorbed IgG and IgG to soluble fibronectin. Both antibodies showed immunoreactive microfibrils in the extracellular matrix of cells in log phase. However, with increasing time in culture, as the cells reached confluence, the immunofluorescence of microfibrils reacting with the absorbed IgG became less intense, whereas that of microfibrils reacting with IgG to fibronectin increased; in confluent cells, essentially no staining was detected with the absorbed IgG, and a dense network of intensely stained microfibrils was seen with IgG to fibronectin. Treatment of these cultures with urea led to partial dissociation of the fibronectin and increased visualization of the microfibrils with the absorbed IgG; double-label immunofluorescence showed that both proteins occurred on the same microfibrils. The localization of immunoreactive sites to the extracellular microfibrils was confirmed by immunoelectron microscopy. Nearly quantitative cleavage with CNBr failed to dissociate the antigenically active fragments of fibronectin from the CNBr fragments of the core proteins of the microfibrils. It was concluded that microfibrils contain core proteins and fibronectin that are codistributed in insoluble, possibly covalently cross-linked, aggregates. The core proteins are first deposited by the cell and, as a function of time in culture, fibronectin gradually coats their surface.

The infrastructure of aortic elastic fibers.

Elastic fibers are composed of a central core of elastin that is amorphous and electron-lucent in conventional transmission electron micrographs and peripheral microfibrils. A complex infrastructure within the amorphous elastin of mature rat aorta is made visible by fixation and staining with a glutaraldehyde-ruthenium red mixture in phosphate buffer or osmium-ruthenium red in cacodylate buffer. The infrastructure is composed of at least two interlacing but distinct elastic structural components; a framework of circumferentially orientated microfibrils and a three-dimensional meshwork of filaments that permeate the fiber. The latter resembles a reticulum that has previously been observed in freeze-fractured and negatively stained elastin and attributed to the supramolecular organization of elastin. Microfibrils also extend from the core of the elastic fiber into the surrounding matrix where they appear to function as anchoring fibers. These observations indicate that the elastic properties of the arterial wall are an integrated function of both elastin and microfibrils.

Involvement of membrane glycophorins in human erythrocytes invaded by Plasmodium falciparum.

The extent of participation and changes of glycophorins (GPs) and membrane proteins in the merozite-human erythrocyte interaction during the invasion of Plasmodium falciparum (Pf) were studied by immunoblotting techniques. Polyclonal antisera to alpha GP and to its C-terminal fragment (residues 82-131) as well as M and N specific monoclonal antibodies (MoAbs) were used. We examined the GPs in the parasite-free saponin lysates, saponin pellets and the culture supernatants of the infected erythrocytes in comparison with the mock cultured normal RBC. Excepting the usual GP patterns, a GP with molecular weight of 77.5 K (band 1') existed in the parasitized erythrocytes and their saponin pellets and several GP bands ranging from 28K to 54K were present in saponin pellets when probed with anti-GP and anti-peptide C sera. This reflected the disintegration of erythrocyte membrane alpha GP through the invasion of Pf. The alpha 2 GP in the saponin pellets of the parasitized MM erythrocytes surprisingly cross-reacted with the N MoAb, implying that it may have come from the intermingling of the host alpha GP in MM erythrocytes with the parasite alpha GP reacting to N MoAb. The saponin pellets of parasitized erythrocytes preserved a considerable amount of ankyrin, band 3, protein 4.1 and 4.2, while the GP bands were densely mixed with the parasite proteins and the disintegrated products of membrane proteins. Four erythrocyte-binding antigens (EBA) of 143 K, 135 K, 115 K and 107 K recognized by malaria hyperimmune serum were detected in the culture supernatants of Pf, some of them appeared in the infected erythrocytes, their saponin lysates and pellets.

Molecular genetics of glycophorin MNS variants.

The antigens for the MNS blood group system are Glycophorins A and B (GPA,GPB), products of the GPA gene family. The existence of close to 40 variant phenotypes of this blood group system has been documented by serological analyses. Here is summarized the molecular basis for a large number of variants, including all the variants of the Miltenberger complex and several isoforms of Sta; also, Dantu, Sat, He, Mg, and deletion variants Ena, S-s-U- and Mk. The diversity is based predominantly on gene recombinations, namely unequal homologous recombinations and/or gene conversions, often coupled to pre-mRNA splicing. Most rearrangements occurred between GPA and GPB alleles, and were confined to hot-spots within the 4 kb region coding for the extracellular domain. The homologous region in GPE, the third member of the gene family, was involved only rarely. Sites of the variant epitopes are mapped to new intra- and inter-exon junctions or to patches of previously silenced sequences that become expressed following recombination.

Characterization of a genomic hybrid specifying the human erythrocyte antigen Dantu: Dantu gene is duplicated and linked to a delta glycophorin gene deletion.

Dantu is a rare antigen of the human MNSs blood group system carried by a hybrid glycophorin on the erythrocyte membrane. To delineate its structure and relationship to alpha and delta glycophorins at the genomic level, we analyzed DNA from a three-generation family, in which both the presence of Dantu and Mi-III (another rare MNSs antigen) and the absence of delta glycophorin were seen. Three gross alterations in the gene cluster correlated with the observed phenotypes. Differential hybridization, secondary restriction analysis, and sequence-specific oligonucleotide probing established that Dantu glycophorin is encoded by a distinct hybrid gene derived from fusion of delta and alpha genes. The junction point of the Dantu gene was assigned to the region spanning codons 31-39 of delta and codons 72-79 of alpha genes. Dosage quantitation suggested that Dantu gene is duplicated and tandemly arranged. Identification of delta gene deletion disclosed the molecular basis for the absence of delta glycophorin in two Dantu and Mi-III double heterozygotes. The DNA inheritance pattern together with DNA typing of MN blood groups verified the genotypes and established that the duplicated Dantu hybrid gene is linked to alpha M gene as well as a delta gene deletion. The origin of such a haplotype in a segment of Black population would require two recombination events via either unequal homologous crossing-over or gene conversion.

Structural polymorphism within the amino-terminal region of MM, NN, and MN glycoproteins (glycophorins) of the human erythrocyte membrane.

MM, NN, and MN glycoproteins of human erythrocytes from single donors were cleaved by cyanogen bromide into three fragments-A, B, and C-which, upon gel electrophoresis, appeared to be common to the three antigens. Phenol/aqueous urea partitioning and gel filtration were used to separate the peptides quantitatively. Peptide C lacked carbohydrate and homoserine and represented the carboxyl-terminal portion of the glycoproteins. Peptides A and B contained one homoserine each and accounted for all the carbohydrate of the glycoproteins. The peptide portion of glycopeptide A from MM, NN, or MN antigens consisted of eight amino acid residues, of which six were homologous and two varied according to blood type. The variants were serine and glycine in glycopeptide A(MM), leucine and glutamic acid in A(NN), and half-residues of serine, glycine, leucine, and glutamic acid in A(MN). Serine was the amino-terminal residue in A(MM), leucine in A(NN), and one half residue of serine and leucine in A(MN). Each glycopeptide carried two tetrasaccharides (2 NANA, 1 Gal, 1 GalNAc) and one trisaccharide (NANA, Gal, GalNAc) linked O-glycosidically to one serine and two threonines as determined by beta-elimination and sulfite addition. The carbohydrate units were attached to serine and threonine located in the invariant region, because the amino-terminal serine residue could be oxidized by periodate. The M-N antigens are believed to be products of allelic genes which are expressed exclusively in homozygotes and equimolarly in heterozygotes.

Multiple origins of the human glycophorin Sta gene. Identification of hot spots for independent unequal homologous recombinations.

Human glycophorin Sta (HGpSta), one of the structural variants of erythrocyte membrane sialoglycoproteins, is encoded by a delta-alpha hybrid gene that arose from a single unequal crossover between the parent HGpB(delta) and HGpA(alpha) genes. We report here the identification of two new HGpSta genes (type A and type B) in four unrelated Sta heterozygotes from two ethnic groups. These Sta genes represent distinct genetic isoforms that differ from the previously reported Sta gene (type C) in the location of crossing-over sites. Comparison of nucleotide sequences among HGpB(delta), HGpA(alpha), and HGpSta type A genes revealed that the delta-alpha unequal crossover for the Sta type A gene occurred 110-246 base pairs downstream from pseudoexon III. In the crossing-over site of this Sta gene, an AT-rich sequence lying 3' to a nonameric palindrome was found to be highly similar to the lambda phage attachment site, att B, in inverted orientation. In the Sta type B gene, the delta-alpha crossing-over point was localized to an AG-rich sequence that is 302-490 base pairs downstream from pseudoexon III. Multiple lambda chi-like elements were identified at the crossover boundaries and within the breakpoint of this Sta gene. These results suggest strongly that recurrent and independent unequal recombination events have occurred in the formation of multiple Sta genes and that particular genomic sequences are important in defining the recombination sites for these homology-driven processes.

Identification of recombination events resulting in three hybrid genes encoding human MiV, MiV(J.L.), and Sta glycophorins.

MiV, MiV(J.L.), and Sta glycophorins specify the respective variant phenotypes of the human MNSs blood group system. We report that unequal but homologous crossing-over between alpha and delta glycophorin genes results in three hybrid genes encoding MiV, MiV(J.L.), and Sta glycophorins. Restriction mapping and allele-specific oligonucleotide hybridization grossly defined the third intron as the probable crossing-over site and showed that MiV and MiV(J.L.) genes are arranged in the same 5' alpha-delta 3' frame whereas Sta gene is in a reciprocal 5'delta-alpha 3' configuration. Genomic sequences spanning the extracellular domain exons 2 to 4 were amplified from each variant gene by polymerase chain reaction and determined by direct DNA sequencing. Comparison of nucleotide sequences encompassing the third intron showed that the three hybrid genes differed in location of crossing-over sites. The alpha-delta breakpoints in MiV and MiV(J.L.) genes were localized to the 3' end of the HindIII site downstream from exon 3 and to the 5' end immediately upstream from exon 4, respectively, whereas the delta-alpha breakpoint in Sta gene resided in between. An AAAGT sequence oriented in either forward or reverse direction was identified within the crossing-over region of each hybrid gene whose surrounding sequences bear a strong local strand asymmetry. The single nucleotide substitution in exon 4 of MiV and MiV(J.L.) genes (ACG [Thr] to ATG [Met]) demonstrated that the two genes differed in the delta glycophorin alleles that must have participated in the recombination.

Molecular genetics of human erythrocyte MiIII and MiVI glycophorins. Use of a pseudoexon in construction of two delta-alpha-delta hybrid genes resulting in antigenic diversification.

Human glycophorins alpha and delta (or A and B) specify the MNSs blood group antigens; they exhibit considerable structural variation among populations. We show that two variant phenotypes of Miltenberger class III and VI are encoded by similar hybrid glycophorin genes in a delta-alpha-delta arrangement. Restriction mapping identified altered fragments unique to the MiIII and MiVI genes. Genomic sequences spanning exons 2 to 4 of the two genes were obtained by allele-specific polymerase chain reaction. Restriction analysis and direct sequencing of the amplified DNA revealed that MiIII and MiVI genes are identical to the delta gene except that, in both, an internal segment of the delta gene has been replaced by its homologous counterpart of the alpha gene, resulting in a delta-alpha-delta hybrid structure. In the process of hybrid formation a portion of alpha exon 3 and intron 3, that carries a functional 5' splicing signal, has been fused to an exon-like sequence in the delta gene that retains a 3' but lacks a 5' splicing signal. These rearrangements created a composite exon resulting in the expression of the ordinarily unexpressed delta gene sequence and conferred the hybrid proteins with new antigenic specificities. The expression of this sequence in MiIII glycophorin is directly demonstrated by protein sequencing. MiIII and MiVI genes differ in the location of upstream (delta-alpha) and downstream (alpha-delta) breakpoints and in the length of sequence replacement. The delta-alpha breakpoints of the two genes occur at different locations within a 35-base pair sequence of exon 3 that is clustered with multiple inverted repeats, whereas the alpha-delta breakpoints reside downstream in two dissimilar blocks of sequences of intron 3. The minimal length of the delta gene sequence that has been replaced by the alpha gene is 55 base pairs in the MiIII gene and 131 base pairs in the MiVI gene. Such segmental DNA transfers may have proceeded unidirectionally through the mechanisms of gene conversion.

Molecular genetics of the glycophorin gene family, the antigens for MNSs blood groups: multiple gene rearrangements and modulation of splice site usage result in extensive diversification.

The purpose of the review is to describe a system of human erythrocyte membrane glycoproteins exhibiting extensive diversity. Glycophorins A and B (GPA and GPB) are the antigens of the MNSs blood groups; thus individuals bearing variant glycophorins can be readily identified by serological typing. Examination of the wide array of variants of these antigens showed that they include many forms, possibly made evident by lack of constraints due to the apparent dispensability of the parent molecules. This article reviews the molecular genetics of 25 variants of the glycophorin gene family, whose common denominator is that they arise from unequal gene recombinations or gene conversions coupled to splice-site mutations. Most rearrangements occurred within a 2-kb region mainly within GPA and GPB of the gene family and only rarely within the third member, GPE. The key feature is the shuffling of sequences within two specific exons (one of which is silent), homologous in the two parent genes. This has resulted in expression of a mosaic of sequences within this region, leading to polymorphism. The common pattern of recombinations coupled to pre-mRNA splicing was the predominant mechanism of the origin of glycophorin diversity. Thus far this mechanism appears to be unique among human gene families. It could have occurred by chance rearrangements among closely linked genes and been driven by a biological advantage, not as yet identified. This remains to be established. Nevertheless, gene rearrangements observed here are akin to those reported for the major histocompatibility complex (MHC). In the glycophorin family the small size of the region within which gene interactions have occurred and the participation of essentially only two alleles makes this relatively simpler system more focused and easier to dissect and describe molecularly.

Glycophorins of human erythroleukemic K562 cells.

Glycophorins related to alpha glycophorin, of the human erythrocyte membrane, were isolated from human erythroleukemic K562 cells. The glycophorins were purified using sodium dodecyl sulfate (SDS)/trichloroacetic acid fractionation and Folch and hot phenol extractions. 0.1-0.2 micrograms was obtained/10(8) cells, or approximately a 15% yield. SDS-gel electrophoresis revealed a pattern similar to erythrocyte alpha glycophorin except for the slower mobility of the glycophorin monomer. Two populations of K562 glycophorins, present in nearly equivalent amounts, were distinguished by their binding to Lens culinaris lectin agarose. The two populations exhibited similar gel electrophoretic patterns except for the presence of delta-like glycophorin exclusively in the population that did not bind to L. culinaris lectin. Immunoblotting revealed a lack of reaction of the major alpha and delta-like glycophorin bands in all K562 glycophorins with M or N erythrocyte glycophorin-specific monoclonal antibodies. Only minor species of intermediate electrophoretic mobility in glycophorins not binding to L. culinaris showed a reaction with these antibodies. Both populations of glycophorins incorporated radiolabeled glucosamine, mannose, and fucose and contained O-glycosidically linked tri- and tetrasaccharides, present in a ratio of approximately 1:1 indicating a significant degree of hyposialylation when compared to erythrocyte alpha glycophorin. No precursor/product relationship was demonstrated between the major forms of two populations. K562 cell surface labeling with lactoperoxidase revealed that only the glycophorins that exhibited binding to L. culinaris were accessible to iodination and could be the only species expressed at the cell surface.