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Biomedical subjects

J F Bateman

Publications and source records attributed to J F Bateman.

At least 37 records · Page 2Linked to original sources
PubMed1998

The role of the alpha3(VI) chain in collagen VI assembly. Expression of an alpha3(VI) chain lacking N-terminal modules N10-N7 restores collagen VI assembly, secretion, and matrix deposition in an alpha3(VI)-deficient cell line.

Collagen VI is a microfibrillar protein found in the extracellular matrix of virtually all connective tissues. Three genetically distinct subunits, the alpha1(VI), alpha2(VI), and alpha3(VI) chains, associate intracellularly to form triple-helical monomers, which then assemble into disulfide-bonded dimers and tetramers before secretion. Although sequence considerations suggest that collagen VI monomers composed of all three chains are the most stable isoform, the precise chain composition of collagen VI remains controversial and alternative assemblies containing only alpha1(VI) and alpha2(VI) chains have also been proposed. To address this question directly and study the role of the alpha3(VI) chain in assembly, we have characterized collagen VI biosynthesis and in vitro matrix formation by a human osteosarcoma cell line (SaOS-2) that is deficient in alpha3(VI) production. Northern analysis showed an abundance of alpha1(VI) and alpha2(VI) mRNAs, but no detectable alpha3(VI) mRNA was apparent in SaOS-2 cells. By day 30 of culture, however, small amounts of alpha3(VI) mRNA were detected, although the level of expression was still much less than alpha1(VI) and alpha2(VI). Collagen VI protein was not detected in SaOS-2 medium or cell layer samples until day 30 of culture, demonstrating that despite the abundant synthesis of alpha1(VI) and alpha2(VI), no stable collagen VI protein was produced without expression of alpha3(VI). The alpha1(VI) and alpha2(VI) chains produced in the absence of alpha3(VI) were non-helical and were largely retained intracellularly and degraded. The critical role of the alpha3(VI) chain in collagen VI assembly was directly demonstrated after stable transfection of SaOS-2 cells with an alpha3(VI) cDNA expression construct that lacked 4 of the 10 N-terminal type A subdomains. The transfected alpha3(VI) N6-C5 chains associated with endogenous alpha1(VI) and alpha2(VI) and formed collagen VI dimers and tetramers, which were secreted and deposited into an extensive network in the extracellular matrix. These data demonstrated that alpha3(VI) is essential for the formation of stable collagen VI molecules and subdomains N10-N7 are not required for molecular assembly.

Ascorbic Acid↗
PubMed1998

Reduced collagen VI causes Bethlem myopathy: a heterozygous COL6A1 nonsense mutation results in mRNA decay and functional haploinsufficiency.

We have identified a new pathogenic mechanism for an inherited muscular dystrophy in which functional haploinsufficiency of the extracellular matrix protein collagen VI causes Bethlem myopathy. The heterozygous COL6A1 mutation results in a single base deletion from the mRNA and a premature stop codon. The mutant mRNA is unstable, subject to nonsense-mediated mRNA decay, and is almost completely absent both from patient fibroblasts and skeletal muscle, resulting in haploinsufficiency of the alpha1(VI) subunit and reduced production of structurally normal collagen VI. This is the first example of a muscular dystrophy caused by haploinsufficiency of a structural protein or member of the dystrophin-glycoprotein complex, and identifies collagen VI as a critical contributor to cell-matrix adhesion in skeletal muscle.

Cells, Cultured↗
PubMed1998

Effects of relaxin, pregnancy and parturition on collagen metabolism in the rat pubic symphysis.

The aim of this study was to examine the changes in collagen metabolism that occur during pregnancy and parturition and upon relaxin administration to the rat pubic symphysial interpubic tissue. Pubic symphyses were collected from non-pregnant, and intact and ovariectomised pregnant Sprague-Dawley rats at days 15, 18 and 21 of pregnancy as well as during and after delivery, and analysed for collagen content and solubility. SDS-PAGE was used to determine collagen composition. During pregnancy and particularly during birth, there was a significant reduction in both the tissue wet (57+/-3%) and dry (43+/-3%) weight (n=7), which coincided with a significant increase in water content (to 80%) and was attributed to a significant (P<0.05) reduction in overall tissue collagen content (by 47+/-2%). This resulted in both soluble (10%) and insoluble (90%) collagen levels being reduced, but gel electrophoresis demonstrated the presence of types I, II and V collagen in all samples. Western blot analysis confirmed the presence of type II collagen throughout pregnancy, confirming that the rat pubic symphysis remained a fibrocartilaginous tissue throughout gestation. In the absence of the ovaries and hence relaxin, tissue collagen content and solubility were not significantly different from control measurements. However, tissues of ovariectomised animals treated with oestrogen and progesterone (pellets) and relaxin (injection) contained collagen levels that mimicked those of late pregnancy and parturition. These results suggest that relaxin plays an important role in regulating collagen catabolism during gestation in the rat.

Analysis of Variance↗
PubMed1997

In vitro expression analysis of collagen biosynthesis and assembly.

While the generalised pathway of collagen biosynthesis is well understood, the specific molecular interactions that drive chain recognition and assembly and the formation of tissue-specific extracellular supramolecular structures have not been elucidated. This review focuses on the use of in vitro collagen expression systems to explore some of these fundamental questions on the molecular basis of normal and mutant collagen assembly. Three in vitro expression/assembly systems are discussed. Firstly, a simple cell-free transcription/translation system to study the initial stages of collagen chain assembly. Secondly, a novel T7-driven high level expression system, using a recombinant vaccinia virus expressing T7 RNA polymerase, in transiently transfected cells which allows appropriate postranslational modification and collagen folding. Thirdly, the more complex questions of normal and mutant collagen extracellular matrix assembly are addressed by stable transfection and expression in cells which allow the formation of a 'tissue equivalent' matrix during long-term culture.

Animals↗
PubMed1996

Clinicopathologic findings in congenital aneurysms of the great vessels.

We describe the clinical, histopathologic, and angiographic findings in four children with congenital abnormalities of the great vessels of unknown cause, comprising either single or multiple arterial aneurysms, aortic/arterial dilatation, vessel tortuosity, or combinations of these abnormalities. Two children had early and severe respiratory distress due to aneurysmal compression of the trachea. All children had diffuse dilatation of several arteries, and two children also had tortuosity of multiple arteries. Progression of these abnormalities was clearly evident in one child, in whom diffuse vessel irregularity and tortuosity affected intra-abdominal, and intra and extra-cranial arteries. One child died at 5 years, while the other three have undergone successful surgical repair in the first 3 months of life and are now well, between age 2.5 and 7 years. The phenotype of each child appears unique but all have in common the rare finding of aneurysms of the aorta and main pulmonary artery. Congenital aortic aneurysms did not occur as an isolated finding in any of these children.

Abnormalities, Multiple↗
PubMed1996

Multiexon deletions in the type I collagen COL1A2 gene in osteogenesis imperfecta type IB. Molecules containing the shortened alpha2(I) chains show differential incorporation into the bone and skin extracellular matrix.

Osteogenesis imperfecta (OI) type IB is a rare subset of the mildest form of OI, clinically characterized by moderate bone fragility, blue sclera, and dentinogenesis imperfecta. Cultured skin fibroblasts from two unrelated individuals (OI-197 and OI-165) with the typical features of OI type IB produced shortened alpha2(I) chains. Reverse transcription-polymerase chain reaction of the alpha2(I)-cDNA revealed deletions in the triple helical domain of 5 exons (exons 7-11) in OI-197, and 8 exons (exons 10-17) in OI-165. This exon skipping was caused by genomic deletions in one allele of COL1A2 with the breakpoints located in introns 6 and 11 in OI-197, and introns 9 and 17 in OI-165. The secretion and deposition of the mutant collagen into the matrix was measured in vitro in cultures of skin fibroblasts and bone osteoblasts, grown in the presence of ascorbic acid to induce collagen matrix formation and maturation, as well as in collagen extracts from skin and bone. The secretion of mutant collagen was impaired and long term cultures of fibroblasts showed that the mutant collagen was not incorporated into the mature collagenous matrix produced in vitro by skin fibroblasts from both patients. Likewise, the shortened alpha2(I) chain was not demonstrable in skin extracts. In contrast, bone extracts from OI-197 showed the presence of the mutant collagen. This incorporation of the abnormal collagen into the mature matrix was also demonstrated in long term cultures of the patient's osteoblasts. The deposition of the mutant collagen by bone osteoblasts but not by skin fibroblasts demonstrates a tissue specificity in the incorporation of mutant collagen into the matrix which may explain the primary involvement of bone and not skin in these patients.

Adolescent↗
PubMed1996

Site-directed mutagenesis of human type X collagen. Expression of alpha1(X) NC1, NC2, and helical mutations in vitro and in transfected cells.

Type X collagen is a short chain collagen expressed in the hypertrophic zone of calcifying cartilage during skeletal development and bone growth. The alpha1(X) homotrimer consists of three protein domains, a short triple helix (COL1) flanked by nonhelical amino-terminal (NC2) and carboxyl-terminal (NC1) domains. While mutations of the NC1 domain result in Schmid metaphyseal chondrodysplasia, which suggests a critical role for this protein domain, little biochemical detail is known about type X collagen synthesis, secretion, and the mechanisms of molecular assembly. To study these processes, a range of mutations were produced in human alpha1(X) cDNA and the biochemical consequences determined by in vitro expression, using T7-driven coupled transcription and translation, and by transient transfection of cells. Three NC1 mutants, which were designed to be analogous to Schmid mutations (1952delC, 1963del10, and Y598D), were unable to assemble into type X collagen homotrimers in vitro, but the mutant chains did not associate with, or interfere with, the efficiency of normal chain assembly in co-translations with a normal construct. Expression in transiently transfected cells confirmed that mutant type X collagen assembly was also compromised in vivo. The mutant chains were not secreted from the cells but did not accumulate intracellularly, suggesting that the unassociated mutant chains were rapidly degraded. In-frame deletions within the helix (amino acid residues 72-354) and the NC2 domain (amino acid residues 21-54) were also produced. In contrast to the NC1 mutations, these mutations did not prevent assembly. Mutant homotrimers and mutant-normal heterotrimers were formed in vitro, and the mutant homotrimers formed in transiently transfected cells had assembled into pepsin-stable triple helical molecules which were secreted.

Animals↗
PubMed1996

An alpha 1(II) Gly913 to Cys substitution prevents the matrix incorporation of type II collagen which is replaced with type I and III collagens in cartilage from a patient with hypochondrogenesis.

A heterozygous mutation in the COL2A1 gene was identified in a patient with hypochondrogenesis. The mutation was a single nucleotide transition of G3285T that resulted in an amino acid substitution of Cys for Gly913 in the alpha 1(II) chain of type II collagen. This amino acid change disrupted the obligatory Gly-X-Y triplet motif required for the normal formation of a stable collagen triple helix and prevented the deposition of type II collagen into the proposita's cartilage, which contained predominantly type I and III collagens and minor amounts of type XI collagen. Biosynthetic analysis of collagens produced and secreted by the patient's chondrocytes cultured in alginate beads was consistent with the in vivo matrix composition, demonstrating that the main products were type I and III collagens, along with type XI collagen. The synthesis of the cartilage-specific type XI collagen at similar levels to controls indicated that the isolated cartilage cells had re-differentiated to the chondrocyte phenotype. The chondrocytes also produced small amounts of type II collagen, but this was post-translationally overmodified and not secreted. These data further delineate the biochemical and phenotypic consequences of mutations in the COL2A1 gene and suggest that cartilage formation and bone development can take place in the absence of type II collagen.

Adult↗
PubMed1996

The phenotypic features of osteogenesis imperfecta resulting from a mutation of the carboxyl-terminal pro alpha 1 (I) propeptide that impairs the assembly of type I procollagen and formation of the extracellular matrix.

The features of a baby with lethal perinatal osteogenesis imperfecta (OI-II), resulting from the substitution of tryptophan 94 by cysteine in the carboxyl-terminal propeptide of pro alpha 1 (I) chains of type I procollagen, were studied. The limbs and torso were of normal length, shape, and proportion. Similarly, all bones were of relatively normal shape and the long bones showed normal metaphyseal modelling. These clinical and radiographic features were similar to those observed in another baby with OI-II resulting from a mutation of the carboxy-terminal propeptide of pro alpha 1 (I) chains but dissimilar from those reported in babies with OI-II resulting from helical mutations of type I collagen.

Bone and Bones↗
PubMed1996

Disrupted growth plates and progressive deformities in osteogenesis imperfecta as a result of the substitution of glycine 585 by valine in the alpha 2 (I) chain of type I collagen.

The skeleton of a child with osteogenesis imperfecta type III, resulting from the substitution of glycine 586 by valine in the triple helical domain of the alpha 2 (I) chain of type I collagen, was severely porotic but contained lamellar bone and Haversian systems. From early childhood, structural failure of the bone resulted in the disruption of growth plates, progressive bone deformities, and severe growth retardation.

Adolescent↗
PubMed1996

The effect of relaxin on collagen metabolism in the nonpregnant rat pubic symphysis: the influence of estrogen and progesterone in regulating relaxin activity.

The aim of this study was to examine the effects of relaxin on collagen content, solubility, and composition in the rat pubic symphysis. Nonpregnant, female Sprague-Dawley rats were bilaterally ovariectomized and either unprimed or primed with estrogen or progesterone alone, or a combination of estrogen and progesterone. One week later these animals were given increasing doses of a synthetic human (gene-2) relaxin (0-100 micrograms) before being killed 16 h later. Their pubic symphysial tissues were then removed and analyzed for collagen content and solubility, whereas collagen composition was determined by SDS-PAGE. Relaxin administration significantly increased the length (140 +/- 6%) and weight (170 +/- 9%) of the interpubic fibrocartilage in estrogen-primed rats (n = 15). At the same time, it decreased the total collagen content by 68 +/- 6%, without altering the proportions of collagen types, which were predominantly type I (85%) and type II collagen (15%). Relaxin administered alone reduced the total collagen content by 64 +/- 4% but had no effect on collagen solubility or composition. Progesterone abolished the effects of relaxin in estrogen-primed rats. It is concluded that relaxin has a potent effect on the amount of collagen in the rat pubic symphysis that is enhanced by estrogen and antagonized by progesterone. The changes in the extracellular matrix within the pubic symphysis induced by relaxin may be important in the modifications that this tissue undergoes during pregnancy.

Animals↗
PubMed1995

Abnormal type III collagen produced by an exon-17-skipping mutation of the COL3A1 gene in Ehlers-Danlos syndrome type IV is not incorporated into the extracellular matrix.

A novel heterozygous mutation of the COL3A1 gene that encodes the alpha 1(III) chains of type III collagen was identified in a family with the acrogeric form of Ehlers-Danlos syndrome type IV (EDS-IV). Cultured dermal fibroblasts produced normal and shortened alpha 1(III) chains. The triple helix of the latter chain was shortened owing to a 33 amino acid deletion of Gly-184 to Pro-216. The corresponding region of cDNA lacked 99 base pairs from nucleotides 1051 to 1149. The deletions corresponded exactly to the normal sequence encoded by exon 17 of the COL3A1 gene. The proband was heterozygous for a T to G transversion at position +2 of intron 17, which resulted in skipping of exon 17. The splicing defect was not corrected by growing the fibroblasts at 33 degrees C and no other splicing variants were identified at 33 or 37 degrees C. The affected brother had the same mutation but his unaffected mother did not. Heterotrimeric type III collagen molecules containing normal and mutant chains were retained within the cell. The mutant homotrimeric molecules were modified and secreted normally and were thermally stable. These normal characteristics of the mutant homotrimers suggested that the loss of ten Gly-Xaa-Yaa triplets (where Gly-Xaa-Yaa is a repetitive amino acid triplet structure in which Xaa and Yaa are other amino acids, proline and hydroxyproline being more common in the Yaa position) did not adversely affect the formation and stability of the triple helix or the structural requirements for secretion. However, the mutant homotrimers were not incorporated into the extracellular matrix of an in vitro model of EDS-IV dermis. The EDS-IV phenotype in this family was probably due to a deficiency in the amount of normal type III collagen available for formation of the heterotypic collagen fibrils of the extracellular matrix. Intracellular and extracellular quality-control mechanisms prevented the incorporation of heterotrimeric and homotrimeric mutant type III collagen molecules into the cross-linked extracellular matrix.

Adult↗
PubMed1995

The type I collagen pro alpha 1(I) COOH-terminal propeptide N-linked oligosaccharide. Functional analysis by site-directed mutagenesis.

The C-propeptides of the pro alpha 1(I) and pro alpha 2(I) chains of type I collagen are each substituted with a single high-mannose N-linked oligosaccharide. Conservation of this motif among the fibrillar collagens has led to the proposal that the oligosaccharide has structural or functional importance, but a role in collagen biosynthesis has not been unambiguously defined. To examine directly the function of the pro alpha 1(I) C-propeptide N-linked oligosaccharide, the acceptor Asn residue was changed to Gln by site-directed mutagenesis. In transfected mouse Mov13 and 3T6 cells, unglycosylated mutant pro alpha 1(I) folded and assembled normally into trimeric molecules with pro alpha 2(I). In biosynthetic pulse-chase experiments mutant pro alpha 1(I) were secreted at the same rate as wild-type chains; however, following secretion, the chains were partitioned differently between the cell layer and medium, with a greater proportion of the mutant pro alpha 1(I) being released into the medium. This distribution difference was not eliminated by the inclusion of yeast mannan indicating that the high-mannose oligosaccharide itself was not binding to the matrix or the fibroblast surface after secretion. Subtle alterations in the tertiary structure of unglycosylated C-propeptides may have decreased their affinity for a cell-surface component. Further support for a small conformational change in the mutant C-propeptides came from experiments suggesting that unglycosylated pro alpha 1(I) chains were cleaved in vitro by the purified C-proteinase slightly less efficiently than wild-type chains. Mutant and normal pro alpha 1(I) were deposited with equal efficiency into the 3T6 cell accumulated matrix, thus the reduced cleavage by C-proteinase and altered distribution in the short pulse-chase experiments were not functionally significant in this in vitro extracellular matrix model system.

Amino Acid Sequence↗
PubMed1995

Distinct skeletal abnormalities in four girls with Shprintzen-Goldberg syndrome.

We describe 4 girls with Shprintzen-Goldberg syndrome. Skeletal abnormalities common to 3 of them include bowing of long bones (with a variable degree of progression over time), flare of the metaphyses, a large anterior fontanel with persistent patency into the second to fourth years of life, 13 pairs of ribs, distinct vertebral abnormalities which were absent neonatally but evolved by the second year of life, and progressive osteopenia. These abnormalities were generalized and, in one case, progressive over the first few years of life. Communicating hydrocephalus was present in all 4 cases. The eldest, an 11-year-old girl, had additional anomalies not reported previously in this syndrome, including intestinal malrotation, an anteriorly placed anus, and mild cerebral atrophy. This is the first detailed report of skeletal manifestations in this rare disorder of unknown cause. These cases, in conjunction with a review of the literature, suggest that skeletal abnormalities are common in Shprintzen-Goldberg syndrome.

Abnormalities, Multiple↗
PubMed1995

Endoplasmic reticulum-mediated quality control of type I collagen production by cells from osteogenesis imperfecta patients with mutations in the pro alpha 1 (I) chain carboxyl-terminal propeptide which impair subunit assembly.

A heterozygous single base change in exon 49 of COL1A1, which converted the codon for pro alpha 1(I) carboxyl-terminal propeptide residue 94 from tryptophan (TGG) to cysteine (TGT) was identified in a baby with lethal osteogenesis imperfecta (OI64). The C-propeptide mutations in OI64 and in another lethal osteogenesis imperfecta cell strain (OI26), which has a frameshift mutation altering the sequence of the carboxyl-terminal half of the propeptide (Bateman, J. F., Lamande, S. R., Dahl, H.-H. M., Chan, D., Mascara, T. and Cole, W. G. (1989) J. Biol. Chem. 264, 10960-10964), disturbed procollagen folding and retarded the formation of disulfide-linked trimers. Although assembly was delayed, the presence of slowly migrating, overmodified alpha 1(I) and alpha 2(I) chains indicated that mutant pro alpha 1(I) could associate with normal pro alpha 1(I) and pro alpha 2(I) to form pepsin-resistant triple-helical molecules, a proportion of which were secreted. Further evidence of the aberrant folding of mutant procollagen in OI64 and OI26 was provided by experiments demonstrating that the endoplasmic reticulum resident molecular chaperone BiP, which binds to malfolded proteins, was specifically bound to type I procollagen and was coimmunoprecipitated in the osteogenesis imperfecta cells but not control cells. Experiments with brefeldin A, which inhibits protein export from the endoplasmic reticulum, demonstrated that unassembled mutant pro alpha 1(I) chains were selectively degraded within the endoplasmic reticulum resulting in reduced collagen production by the osteogenesis imperfecta cells. This biosynthetic deficiency was reflected in the inability of OI64 and OI26 cells to produce a substantial in vitro collagenous matrix when grown in the continuous presence of ascorbic acid to allow collagen matrix formation. Both these carboxyl-terminal propeptide mutants showed a marked reduction in collagen accumulation to 20% (or less) of control cultures, comparable to the reduced collagen content of tissues from OI26.

Base Sequence↗
PubMed1995

Type X collagen multimer assembly in vitro is prevented by a Gly618 to Val mutation in the alpha 1(X) NC1 domain resulting in Schmid metaphyseal chondrodysplasia.

Type X collagen is a homotrimer of alpha 1(X) chains encoded by the COL10A1 gene. It is a highly specialized extracellular matrix component, and its synthesis is restricted to hypertrophic chondrocytes in the calcifying cartilage of the growth plate and in zones of secondary ossification. Our studies on a family with Schmid metaphyseal chondrodysplasia demonstrated that the affected individuals were heterozygous for a single base substitution in the COL10A1 gene, which changed the codon GGC for glycine 618 to GTC for valine in the highly conserved region of the carboxyl-terminal NC1 domain and altered the amino acid sequence in the putative oligosaccharide attachment site. Since hypertrophic cartilage tissues or cell cultures were not available to assess the effect of the mutation, an in vitro cDNA expression system was used to study normal and mutant type X collagen biosynthesis and assembly. Full-length cDNA constructs of the normal type X collagen sequence and also cDNA containing the specific Gly to Val NC1 mutation found in the patient were produced and expressed by in vitro transcription and translation. While the control construct produced type X collagen, which formed trimeric collagen monomers and assembled into larger multimeric assemblies, the mutant collagen was unable to form these larger aggregates. These experiments demonstrated that the mutation disturbed type X collagen NC1 domain interaction and assembly, a finding consistent with the abnormal disorganized cartilage growth plate seen in the patient. These studies provide the first evidence of the effect of a type X collagen mutation on protein structure and function and directly demonstrate the critical role of interactions between NC1 domains in the formation of type X collagen multimeric structures in vitro.

Amino Acid Sequence↗