mua-3, a gene required for mechanical tissue integrity in Caenorhabditis elegans, encodes a novel transmembrane protein of epithelial attachment complexes.

Normal locomotion of the nematode Caenorhabditis elegans requires transmission of contractile force through a series of mechanical linkages from the myofibrillar lattice of the body wall muscles, across an intervening extracellular matrix and epithelium (the hypodermis) to the cuticle. Mutations in mua-3 cause a separation of the hypodermis from the cuticle, suggesting this gene is required for maintaining hypodermal-cuticle attachment as the animal grows in size postembryonically. mua-3 encodes a predicted 3,767 amino acid protein with a large extracellular domain, a single transmembrane helix, and a smaller cytoplasmic domain. The extracellular domain contains four distinct protein modules: 5 low density lipoprotein type A, 52 epidermal growth factor, 1 von Willebrand factor A, and 2 sea urchin-enterokinase-agrin modules. MUA-3 localizes to the hypodermal hemidesmosomes and to other sites of mechanically robust transepithelial attachments, including the rectum, vulva, mechanosensory neurons, and excretory duct/pore. In addition, it is shown that MUA-3 colocalizes with cytoplasmic intermediate filaments (IFs) at these sites. Thus, MUA-3 appears to be a protein that links the IF cytoskeleton of nematode epithelia to the cuticle at sites of mechanical stress.

Hemicentin, a conserved extracellular member of the immunoglobulin superfamily, organizes epithelial and other cell attachments into oriented line-shaped junctions.

him-4 mutations cause a novel syndrome of tissue fragility, defective cell migration and chromosome instability in Caenorhabditis elegans. Null mutants have abnormal escape reflex, mispositioning of the vas deferens and uterus, and mitotic chromosome loss and multinucleate cells in the germline. The him-4 gene product, hemicentin, is a conserved extracellular matrix protein with 48 tandem immunoglobulin repeats flanked by novel terminal domains. Secreted from skeletal muscle and gonadal leader cells, hemicentin assembles into fine tracks at specific sites, where it contracts broad regions of cell contact into oriented linear junctions. Some tracks organize hemidesmosomes in the overlying epidermis. Hemicentin tracks facilitate mechanosensory neuron anchorage to the epidermis, gliding of the developing gonad along epithelial basement membranes and germline cellularization.

Conservation and novelty in the evolution of cell adhesion and extracellular matrix genes.

New proteins and modules have been invented throughout evolution. Gene "birth dates" in Caenorhabditis elegans range from the origins of cellular life through adaptation to a soil habitat. Possibly half are "metazoan" genes, having arisen sometime between the yeast-metazoan and nematode-chordate separations. These include basement membrane and cell adhesion molecules implicated in tissue organization. By contrast, epithelial surfaces facing the environment have specialized components invented within the nematode lineage. Moreover, interstitial matrices were likely elaborated within the vertebrate lineage. A strategy for concerted evolution of new gene families, as well as conservation of adaptive genes, may underlie the differences between heterochromatin and euchromatin.

A novel integrin specificity exemplified by binding of the alpha v beta 5 integrin to the basic domain of the HIV Tat protein and vitronectin.

Several studies have addressed the interaction of the HIV Tat protein with the cell surface. Our analysis of the cell attachment-promoting activity of Tat and peptides derived from it revealed that the basic domain of Tat, not the arg-gly-asp (RGD) sequence, is required for cell attachment to Tat. Affinity chromatography with Tat peptides and immunoprecipitation with various anti-integrin antibodies suggest that the vitronectin-binding integrin, alpha v beta 5, is the cell surface protein that binds to the basic domain of Tat. The Tat basic domain contains the sequence RKKRRQRRR. A related sequence, KKQRFRHRNRKG, present in the heparin-binding domain of an alpha v beta 5 ligand, vitronectin, also bound alpha v beta 5 in affinity chromatography and, in combination with an RGD peptide, was an inhibitor of cell attachment to vitronectin. The alpha v beta 5 interaction with these peptides was not solely due to high content of basic amino acids in the ligand sequences; alpha v beta 5 did not bind substantially to peptides consisting entirely of arginine or lysine, whereas a beta 1 integrin did bind to these peptides. The interaction of alpha v beta 5 with Tat is atypical for integrins in that the binding to Tat is divalent cation independent, whereas the binding of the same integrin to an RGD-containing peptide or to vitronectin requires divalent cations. These data define an auxiliary integrin binding specificity for basic amino acid sequences. These basic domain binding sites may function synergistically with the binding sites that recognize RGD or equivalent sequences.

A type I collagen with substitution of a cysteine for glycine-748 in the alpha 1(I) chain copolymerizes with normal type I collagen and can generate fractallike structures.

Type I procollagen was purified from cultured fibroblasts of a proband with a lethal variant of osteogenesis imperfecta. The protein was a mixture of normal procollagen and mutated procollagens containing a substitution of cysteine for glycine in either one pro alpha 1(I) chain or both pro alpha 1(I) chains, some or all of which were disulfide-linked through the cysteine at position alpha 1-748. The procollagen was then examined in a system for generating collagen fibrils de novo by cleavage of the pCcollagen to collagen with procollagen C-proteinase [Kadler et al. (1987) J. Biol. Chem. 262, 15696-15701]. The mutated collagens and normal collagens were found to form copolymers under a variety of experimental conditions. With two preparations of the protein that had a high content of alpha 1(I) chains disulfide-linked through the cysteine alpha 1-748, all the large structures formed had a distinctive, highly branched morphology that met one of the formal criteria for a fractal. Preparations with a lower content of disulfide-linked alpha 1(I) chains formed fibrils that were 4 times the diameter of control fibrils. The formation of copolymers was also demonstrated by the observation that the presence of mutated collagens decreased the rate of incorporation of normal collagen into fibrils. In addition, the solution-phase concentration at equilibrium of mixtures of mutated and normal collagens was 5-10-fold greater than that of normal collagen.(ABSTRACT TRUNCATED AT 250 WORDS)

A mutation in the pro alpha 2(I) gene (COL1A2) for type I procollagen in Ehlers-Danlos syndrome type VII: evidence suggesting that skipping of exon 6 in RNA splicing may be a common cause of the phenotype.

Fibroblasts from a proband with Ehlers-Danlos syndrome type VII synthesized approximately equal amounts of normal and shortened pro alpha 2(I) chains of type I procollagen. Nuclease S1 probe protection experiments with mRNA demonstrated that the pro alpha 2(I) chains were shortened because of a deletion of most or all of the 54 nucleotides in exon 6, the exon that contains codons for the cleavage site for procollagen N-proteinase. Sequencing of genomic clones revealed a single-base mutation that converted the first nucleotide of intron 6 from G to A. Therefore, the mutation was a change, in the -GT-consensus splice site, that produced efficient exon skipping. Allele-specific oligonucleotide hybridizations demonstrated that the proband's mother, father, and brother did not have the mutation. Therefore, the mutation was a sporadic one. Analysis of potential 5' splice sites in the 5' end of intron 6 indicated that none had favorable values by the two commonly employed techniques for evaluating such sites. The proband is the fourth reported proband with Ehlers-Danlos syndrome VII with a single-base mutation that causes skipping of exon 6 in the splicing of RNA from either the COL1A1 gene or COL1A2 gene. No other mutations in the two type I procollagen genes have been found in the syndrome. Therefore, such mutations may be a common cause of the phenotype. The primers developed should be useful in screening for the same or similar mutations causing the disease.

A novel fibronectin receptor with an unexpected subunit composition (alpha v beta 1).

The integrins are a family of heterodimeric cell surface receptors for extracellular matrix molecules. An analysis of integrin subunits expressed by a number of cell lines identified a novel heterodimer. The alpha subunit of this integrin was immunologically and electrophoretically indistinguishable from the vitronectin receptor alpha subunit (alpha v) and the beta subunit was indistinguishable from beta 1. Affinity chromatography experiments and cell adhesion assays indicated that this receptor complex is a new fibronectin receptor. Its unexpected subunit composition demonstrates the importance of the beta subunit in determining the ligand specificity of integrins and suggests that the current integrin classification scheme needs revision.

Type I procollagen: the gene-protein system that harbors most of the mutations causing osteogenesis imperfecta and probably more common heritable disorders of connective tissue.

Recent data from several laboratories have established that most variants of osteogenesis imperfecta (OI) are caused by mutations in the 2 structural genes for type I procollagen. There are 2 general reasons for the large number of mutations in type I procollagen in OI. One reason is that most of the structure of the procollagen monomer is essential for normal biological function of the protein. The second reason is that most of the mutations cause synthesis of structurally altered pro alpha chains of type I procollagen. The deleterious effects of the structurally altered pro alpha chains are then amplified by at least 3 mechanisms. One mechanism is a phenomenon referred to as "procollagen suicide" whereby altered pro alpha chains cause degradation of normal pro alpha chains synthesized by the same cell. Another mechanism involves the fact that many of the structurally altered pro alpha chains prevent normal processing of the N-propeptides of procollagen and persistence of the N-propeptide interferes with normal fibril assembly. A third mechanism is a recently discovered phenomenon in which a substitution of a bulkier amino acid for glycine can cause a kink in the triple helix of the molecule. The kinked collagen, in turn, causes formation of abnormally branched fibrils. Because the deleterious effects of abnormal pro alpha chains are amplified by these 3 mechanisms, most of the mutations are dominant and many are dominant lethal. The conclusion that most variants of OI are caused by mutations in the structural genes for type I procollagen has broad implications for other diseases that affect connective tissue, diseases such as chondrodystrophies, osteoarthritis, and osteoporosis.

Mutations that alter the primary structure of type I procollagen have long-range effects on its cleavage by procollagen N-proteinase.

Type I/II procollagen N-proteinase was partially purified from chick embryos and used to examine the rate of cleavage of a series of purified type I procollagens synthesized by fibroblasts from probands with heritable disorders of connective tissue. The rate of cleavage was normal with procollagen from a proband with osteogenesis imperfecta that was overmodified by posttranslational enzymes. Therefore, posttranslational overmodification of the protein does not in itself alter the rate of cleavage under the conditions of the assay employed. Cleavage of the procollagen, however, was altered in several procollagens with known mutations in primary structure. Two of the procollagens had in-frame deletions of 18 amino acids encoded by exons 11 and 33 of the pro alpha 2(I) gene. In both procollagens, both the pro alpha 1(I) and the pro alpha 2(I) chains were totally resistant to cleavage. With a procollagen in which glycine-907 of the alpha 2(I) chain domain was substituted with aspartate, both pro alpha chains were cleaved but at a markedly decreased rate. The results, therefore, establish that mutations that alter the primary structure of the pro alpha chains of procollagen at sites far removed from the N-proteinase cleavage site can make the protein resistant to cleavage by the enzyme. The long-range effects of in-frame deletions or other changes in amino acid sequence are probably explained by their disruption of the hairpin structure that is formed by each of the three pro alpha chains in the region containing the cleavage site and that is essential for cleavage of the procollagen molecule by N-proteinase.

A substitution of cysteine for glycine 748 of the alpha 1 chain produces a kink at this site in the procollagen I molecule and an altered N-proteinase cleavage site over 225 nm away.

In previous work (Vogel, B. E., Minor, R. R., Freund, M., and Prockop, D. J. (1987) J. Biol. Chem. 262, 14737-14744), we identified a single-base mutation that converted the glycine at position 748 of the alpha 1 chain of type I procollagen to a cysteine in a proband with a lethal variant of osteogenesis imperfecta. In addition to posttranslational overmodification, the abnormal molecules displayed decreased thermal stability and a decreased rate of secretion. An unexplained finding was that procollagen was poorly processed to pCcollagen in postconfluent cultures of skin fibroblasts. Here, we show that the procollagen synthesized by the proband's cells is resistant to cleavage by procollagen N-proteinase, a conformation-sensitive enzyme. Since the only detectable defect in the molecule was the cysteine for glycine substitution, we assembled several space-filling models to try to explain how the structure of the N-proteinase cleavage site can be affected by an amino acid substitution over 700 amino acid residues or 225 nm away. The models incorporated a phase shift of a tripeptide unit in one or both of the alpha 1 chains. The most satisfactory models produced a flexible kink of 30 degrees or 60 degrees at the site of the cysteine substitution. Therefore, we examined the procollagen by electron microscopy. About 25% of the molecules had a kink not seen in control samples, and the kink was at the site of the cysteine substitution.

A point mutation in a type I procollagen gene converts glycine 748 of the alpha 1 chain to cysteine and destabilizes the triple helix in a lethal variant of osteogenesis imperfecta.

Synthesis of type I procollagen was examined in fibroblasts from a proband with a lethal perinatal variant of osteogenesis imperfecta. After trypsin digestion of the type I procollagen, a portion of the alpha 1 (I) chains was recovered as disulfide-linked dimers. Digestion of the protein with vertebrate collagenase and mapping of cyanogen bromide peptides suggested that a new cysteine residue was present between residues 551 and 775 of the alpha 1 (I) chain. Sequencing of cloned cDNAs prepared using mRNA from the proband's fibroblasts demonstrated that some of the clones contained a single base mutation that converted the glycine codon in amino acid position 748 of the alpha 1 (I) chain to a cysteine codon. About 80% of the type I procollagen synthesized by the proband's fibroblasts had a decreased thermal stability. The results, therefore, were consistent with the conclusion that normal pro-alpha 1 (I) chains and pro-alpha 1 (I) chains containing a cysteine residue in the alpha chain domain were synthesized in about equal amounts and incorporated randomly into type I procollagen. However, only about 10% of the alpha 1 (I) chains generated by trypsin digestion were disulfide-linked. Further studies demonstrated a decreased rate of secretion of type I procollagen containing the new cysteine residue and decreased processing of the protein by procollagen N-proteinase in cultures of postconfluent fibroblasts. Both parents were phenotypically normal and their fibroblasts synthesized only normal type I procollagen. Therefore, the mutation in the proband was a sporadic one and is very likely to have caused the connective tissue fragility that produced the lethal phenotype.

The A and B fragments of normal type I procollagen have a similar thermal stability to proteinase digestion but are selectively destabilized by structural mutations.

Previous studies demonstrated that the thermal stability of the procollagen triple helix can be assayed by digesting the protein for short periods with high concentrations of trypsin and chymotrypsin. Here we cleaved human type I procollagen or collagen with vertebrate collagenase to generate A fragments from the three-quarter amino termini and B fragments from the one-quarter carboxy termini of the molecules. The thermal stabilities of the fragments were then assayed by rapid trypsin/chymotrypsin digestion. Both fragments were resistant up to 36 degrees C and completely degraded between 37 degrees C and 39 degrees C. In subsequent experiments the same assay was carried out with type I procollagens synthesized by fibroblasts from two patients with lethal variants of osteogenesis imperfecta. With one, the A fragments were selectively destabilized, an observation consistent with previous data indicating that the mutation in the patient produced a deletion of 84 amino acids from the middle of the alpha 1(I) chain. With procollagen synthesized by fibroblasts from the second patient the B fragments were selectively destabilized, an observation consistent with preliminary data indicating a mutation that alters the primary structure of the carboxy-terminal region of the alpha 1(I) chain. Therefore, the procedures described here present a simple and direct method for locating mutations that destabilize the collagen triple helix.