Unequal Impact of COL1A1 and COL1A2 Variants on Dentinogenesis Imperfecta.

Dentinogenesis imperfecta (DI) is the main orodental manifestation of osteogenesis imperfecta (OI) caused by COL1A1 or COL1A2 heterozygous pathogenic variants. Its prevalence varies according to the studied population. Here, we report the molecular analysis of 81 patients with OI followed at reference centers in Brazil and France presenting COL1A1 or COL1A2 variants. Patients were submitted to clinical and radiographic dental examinations to diagnose the presence of DI. In addition, a systematic literature search and a descriptive statistical analysis were performed to investigate OI/DI phenotype-genotype correlation in a worldwide sample. In our cohort, 50 patients had COL1A1 pathogenic variants, and 31 patients had COL1A2 variants. A total of 25 novel variants were identified. Overall, data from a total of 906 individuals with OI were assessed. Results show that DI was more frequent in severe and moderate OI cases. DI prevalence was also more often associated with COL1A2 (67.6%) than with COL1A1 variants (45.4%) because COL1A2 variants mainly lead to qualitative defects that predispose to DI more than quantitative defects. For the first time, 4 DI hotspots were identified. In addition, we showed that 1) glycine substitution by branched and charged amino acids in the α2(I) chain and 2) substitutions occurring in major ligand binding regions-MLRB2 in α1(I) and MLBR 3 in α2(I)-could significantly predict DI (P < 0.05). The accumulated variant data analysis in this study provides a further basis for increasing our comprehension to better predict the occurrence and severity of DI and appropriate OI patient management.

Possible interference between tissue-non-specific alkaline phosphatase with an Arg54-->Cys substitution and acounterpart with an Asp277-->Ala substitution found in a compound heterozygote associated with severe hypophosphatasia.

Tissue-non-specific alkaline phosphatase (TNSALP) with an Arg(54)-->Cys (R54C) or an Asp(277)-->Ala (D277A)substitution was found in a patient with hypophosphatasia [Henthorn,Raducha, Fedde, Lafferty and Whyte (1992) Proc. Natl. Acad. Sci. U.S.A.89, 9924-9928]. To examine effects of these missense mutations onproperties of TNSALP, the TNSALP mutants were expressed ectopically inCOS-1 cells. The wild-type TNSALP was synthesized as a 66-kDa endo-beta-N-acetylglucosaminidase H (Endo H)-sensitive form, and processed to an 80-kDa mature form, which is anchored to the plasma membrane via glycosylphosphatidylinositol (GPI). Although the mutant proteins were found to be modified by GPI, digestion with phosphatidylinositol-specific phospholipase C, cell-surface biotinylation and immunofluorescence observation demonstrated that the cell-surface appearance of TNSALP (R54C) and TNSALP (D277A) was either almost totally or partially retarded respectively. The 66-kDa Endo H-sensitive band was the only form, and was rapidly degraded in the cells expressing TNSALP (R54C). In contrast with cells expressing TNSALP(R54C), where alkaline phosphatase activity was negligible, significant enzyme activity was detected and, furthermore, the 80-kDa mature form appeared on the surface of the cells expressing TNSALP (D277A). Analysis by sedimentation on sucrose gradients showed that a considerable fraction of newly synthesized TNSALP (R54C) and TNSALP(D277A) formed large aggregates, indicating improper folding and incorrect oligomerization of the mutant enzymes. When co-expressed with TNSALP (R54C), the level of the 80-kDa mature form of TNSALP (D277A)was decreased dramatically, with a concomitant reduction in enzyme activity in the co-transfected cell. These findings suggest that TNSALP(R54C) interferes with folding and assembly of TNSALP (D277A) intrans when expressed in the same cell, thus probably explaining why a compound heterozygote for these mutant alleles developed severe hypophosphatasia.

A general method for rapid purification of soluble versions of glycosylphosphatidylinositol-anchored proteins expressed in insect cells: an application for human tissue-nonspecific alkaline phosphatase.

A soluble form of tissue-nonspecific alkaline phosphatase was purified to apparent homogeneity from the culture media of Sf9 cells which had been infected with recombinant baculoviruses encoding human tissue-nonspecific alkaline phosphatase (TNSALP). To facilitate purification, an oligonucleotide consisting of 6 tandem codons for histidine and a stop codon was engineered into the TNSALP cDNA. The molecular mass of the enzyme purified through a nickel-chelate column was estimated to be 54 kDa by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. That of the native enzyme was 90 kDa as estimated by gel filtration, indicating that the purified soluble TNSALP is dimeric. The enzyme was used for production of antibodies specific for human TNSALP. Immunoblotting analysis showed a single 80-kDa band in the cell homogenate prepared from Saos-2 (human osteosarcoma) cells. However, upon digestion with peptide: N-glycosidase F, the 80-kDa TNSALP of human origin and the soluble enzyme of insect origin migrated to the same position on SDS-polyacrylamide gel, indicating that the size difference between the two enzymes is ascribed to N-linked oligosaccharides. The antibodies prepared against the purified TNSALP were found to be useful also for immunoprecipitation and immunofluorescence studies.

Membrane topology of Alzheimer's disease-related presenilin 1. Evidence for the existence of a molecular species with a seven membrane-spanning and one membrane-embedded structure.

A significant member of early-onset familial type of Alzheimer's disease cases has been shown to be caused by dominant mutations in either of the two genes encoding presenilin 1 (PS1) and presenilin 2 (PS2). These two proteins are highly homologous to each other and have been reported to be mainly localized to the membranes of intracellular compartments such as the endoplasmic reticulum. Information about the membrane topological structures of these proteins is indispensable for understanding their physiological and pathological roles. Although several models have been proposed previously, their precise membrane topologies remain unknown. In this study, we examined this issue in detail by expressing a series of C-terminally deleted PS1 mutants fused to the hydrophilic portion of Escherichia coli leader peptidase in vitro using a reticulocyte lysate in the presence of microsomal membranes. Our results predict that PS1 exists mainly in a seven membrane-spanning structure with its C-terminal end exposed to the luminal space. This was also confirmed by expressing these fusion proteins in cultured cells. We further showed that a ninth hydrophobic segment is tightly bound to the membrane without spanning it. Based on the above observations, we propose a novel "seven membrane-spanning and one membrane-embedded" topological model for presenilins.

Identification of two mutations in human xanthine dehydrogenase gene responsible for classical type I xanthinuria.

Hereditary xanthinuria is classified into three categories. Classical xanthinuria type I lacks only xanthine dehydrogenase activity, while type II and molybdenum cofactor deficiency also lack one or two additional enzyme activities. In the present study, we examined four individuals with classical xanthinuria to discover the cause of the enzyme deficiency at the molecular level. One subject had a C to T base substitution at nucleotide 682 that should cause a CGA (Arg) to TGA (Ter) nonsense substitution at codon 228. The duodenal mucosa from the subject had no xanthine dehydrogenase protein while the mRNA level was not reduced. The two subjects who were siblings with type I xanthinuria were homozygous concerning this mutation, while another subject was found to contain the same mutation in a heterozygous state. The last subject who was also with type I xanthinuria had a deletion of C at nucleotide 2567 in cDNA that should generate a termination codon from nucleotide 2783. This subject was homozygous for the mutation and the level of mRNA in the duodenal mucosa from the subject was not reduced. Thus, in three subjects with type I xanthinuria, the primary genetic defects were confirmed to be in the xanthine dehydrogenase gene.

The structure of chicken liver xanthine dehydrogenase. cDNA cloning and the domain structure.

The amino acid sequence of chicken liver xanthine dehydrogenase (EC 1.1.1.204) was determined by cDNA cloning and partial amino acid sequencing of the purified enzyme. The enzyme consisted of 1358 amino acids with calculated molecular mass of 149,633 Da. In order to compare the structure of the chicken and rat enzymes, limited proteolysis was performed with the purified chicken liver xanthine dehydrogenase. When the enzyme was digested with subtilisin, it was not converted from the NAD-dependent dehydrogenase type to the O2-dependent oxidase type, in contrast with the mammalian enzyme. However, the enzyme was cleaved mainly into three fragments in a manner similar to that for the rat enzyme at pH 8.2 (20, 37, and 84 kDa) and retaining a full complement of redox centers. The cleavage sites were identified by determination of amino-terminal sequences of the produced fragments. It was concluded that the 20-kDa fragment was amino-terminal, the 84-kDa fragment carboxyl-terminal, and the 37-kDa fragment an intermediate portion in the enzyme protein. On the other hand, when the enzyme was digested with the same protease at pH 10.5, the sample contained only the 20- and 84-kDa portions and lacked the 37-kDa portion. The resultant sample possessed xanthine dichlorophenol indophenol reductase activity, indicating that the molybdenum center remained intact. The absorption spectrum showed the sample was very similar to deflavo-enzyme. From these results and sequence analyses, the domain structure of the enzyme is discussed.

Cloning of the cDNA encoding human xanthine dehydrogenase (oxidase): structural analysis of the protein and chromosomal location of the gene.

The primary structure of human xanthine dehydrogenase (hXDH) was determined by cloning and sequence analysis of the cDNAs encoding the enzyme. The nucleotide (nt) sequence has an open reading frame of 3999 nt encoding a protein of 1333 amino acids (aa) with a calculated M(r) of 146,604. The deduced aa sequence of hXDH is homologous to the previously reported rat XDH (rXDH) and Drosophila melanogaster XDH sequences with identities of 90.2 and 52.0%, respectively. The aa residues involved in both the reversible and the irreversible conversion from the dehydrogenase type to the oxidase type of rXDH are completely conserved between the rat and the human enzymes. This implies that the molecular mechanisms of the conversion of hXDH from dehydrogenase to oxidase are common to those of the well-characterized rXDH. Five sequence variations were detected in the isolated cDNA clones. Spot blot hybridization using flow-sorted human chromosome revealed that the hXDH-encoding gene (hXDH) was located on chromosome 2.

Cloning and sequence analysis of a full length cDNA encoding human mitochondrial 3-oxoacyl-CoA thiolase.

The cDNA sequence of human mitochondrial 3-oxoacyl-CoA thiolase was determined. The nucleotide sequence contains an open reading frame of 1191 base pairs and encodes an amino acid sequence of 397 residues which exhibits 86.6% homology with that of the rat enzyme. Northern blot analysis gave a single mRNA species of 1.6 kb in the human liver, fibroblasts and intercostal muscle.

Structural analysis of an extremely long 5'-noncoding region of rat brain argininosuccinate lyase mRNA: presence of multiple B1 repeats and multiple upstream AUG codons, and a possibility of translational control.

The present detailed analysis of the sequence of the extremely long (967 bp) 5'-noncoding region of a rat brain argininosuccinate lyase cDNA clone, reveals several features of interest. Multiple copies of partial and inverted (antisense) B1 repeats and multiple upstream ATG codons are present in the region, which suggests a possibility of translational control of the argininosuccinate lyase gene expression in rat brain.

Functional expression of the alpha 1 subunit of the AMPA-selective glutamate receptor channel, using a baculovirus system.

Using a baculovirus expression vector system, the alpha 1 subunit of the mouse AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate)-selective glutamate receptor channel was expressed in insect Spdoptera frugiperda cells. Binding studies using [3H]AMPA showed that insect cells infected with the recombinant virus expressed approximately 1.8 x 10(5) binding sites per cell on their surface. The ligand binding characteristics of the receptors expressed in insect cells were examined. The baculovirus-insect cell expression system affords high-efficiency expression of the receptor in sufficient amounts to permit structural and functional analyses.

SRH1 protein, the yeast homologue of the 54 kDa subunit of signal recognition particle, is involved in ER translocation of secretory proteins.

The function of the SRH1 product, the yeast homologue of the 54 kDa subunit of the mammalian signal recognition particle, has been analyzed using a galactose dependent mutant of the gene. SRH1 has been placed under control of the GAL1 promoter and introduced into a haploid cell that had its chromosomal SRH1 copy disrupted. This mutant grows normally on galactose medium but slows down the growth about 10 h after transfer to glucose medium. At the same time, precursor forms of secretory proteins, alpha-mating factor and invertase, accumulate in the cells. This result indicates that the SRH1 product is involved in translocation of precursors of secretory proteins across the endoplasmic reticulum membrane in yeast cells.

Proteolytic conversion of xanthine dehydrogenase from the NAD-dependent type to the O2-dependent type. Amino acid sequence of rat liver xanthine dehydrogenase and identification of the cleavage sites of the enzyme protein during irreversible conversion by trypsin.

The primary structure of rat liver xanthine dehydrogenase (EC 1.1.1.204) was determined by sequence analysis of cDNA and purified enzyme. The enzyme consists of 1,319 amino acid residues with a calculated molecular mass of 145,034 Da, including initiation methionine, and is homologous to the previously reported Drosophila melanogaster enzyme (Lee, C. S., Curtis, D., McCarron, M., Love, C., Gray, M., Bender, W., and Chovnick, A. (1987) Genetics 116, 55-66; Keith, T. P., Riley, M. A., Kreitman, M., Lewontin, R. C., Curtis, D., and Chambers, G. (1987) Genetics 116, 67-73) with an identity of 52%. The enzyme exists originally as the NAD-dependent type in a freshly prepared sample. When the purified NAD-dependent type enzyme was digested with trypsin, it cleaved into three fragments with molecular masses of 20, 40, and 85 kDa and was irreversibly converted to the O2-dependent type. Comparison of the amino-terminal sequences of the three peptide fragments with the cDNA-deduced sequence reveals that the 20-, 40-, and 85-kDa peptide fragments correspond residues to 1-184, 185-539, and 540-1319 of the enzyme, respectively. Comparison of the 5'-p-fluorosulfonylbenzoyladenosine-labeled peptide sequence of the chicken enzyme (Nishino, T., and Nishino, T. (1989) J. Biol. Chem. 264, 5468-5473) reveals that the NAD binding site is associated with the 40-kDa fragment portion of the enzyme. Hydropathy analysis around the cysteine residues suggests that the 2Fe/2S sites are associated with the 20-kDa fragment portion of the enzyme.

Isolation of a yeast gene, SRH1, that encodes a homologue of the 54K subunit of mammalian signal recognition particle.

A 1.7 kilobase HindIII fragment of Saccharomyces cerevisiae DNA was cloned by cross-hybridization with the Escherichia coli secY gene. The complete nucleotide sequence of the 2.6 kb fragment of the yeast genomic DNA containing the cross-hybridizing HindIII fragment was determined. The sequence showed no apparent similarity with that of the E. coli secY gene with the exception of a completely matched sequence of 21 bp, but it contained a 1,623 nucleotide open reading frame coding for a protein of 541 amino acids with a calculated Mr of 59,600. The N-terminal portion of 303 residues of the predicted sequence was homologous to the cytosolic domain of the alpha-subunit of the signal recognition particle receptor (SR alpha), including consensus sequence elements for a GTP binding site, whereas the C-terminal portion of 238 residues had an unusual methionine-rich domain containing several repetitive sequences. An mRNA of 2.0 kb was detected on Northern blotting analysis. The predicted sequence was 48% identical with the reported sequences of the 54K subunit of the mammalian signal recognition particle (SRP54) (Romisch K. et al. (1989) Nature 340, 478-483; Bernstein, H.D. et al. (1989) Nature 340, 482-486). We designated this gene as SRH1 (SRP54 homologue). Gene disruption experiments showed that the SRH1 gene product is essential for cell growth.

The NH2-terminal 14-16 amino acids of mitochondrial and bacterial thiolases can direct mature ornithine carbamoyltransferase into mitochondria.

Unlike most mitochondrial matrix proteins, the mitochondrial 3-oxoacyl-CoA thiolase [EC 2.3.1.16] is synthesized with no cleavable presequence and possesses information for mitochondrial targeting and import in the mature protein. This mitochondrial thiolase is homologous with the mature portion of peroxisomal 3-oxoacyl-CoA thiolase and acetoacetyl-CoA thiolase [EC 2.3.1.9] of Zoogloea ramigera along the entire sequence. A hybrid gene encoding the NH2-terminal 16 residues (MALLRGVFIVAAKRTP) of the mitochondrial thiolase fused to the mature portion of rat ornithine carbamoyltransferase [EC 2.1.3.3] (lacking its own presequence) was transfected into COS cells, and subcellular localization of the fusion protein was analyzed. Cell fractionation and immunocytochemical analyses showed that the fusion protein was localized in the mitochondria. These results indicate that the NH2-terminal 16 residues of the mitochondrial thiolase function as a noncleavable signal for mitochondrial targeting and import of this enzyme protein. The fusion protein containing the NH2-terminal 14 residues (MSTPSIVIASARTA) of the bacterial thiolase was also localized in the mitochondria. On the other hand, the fusion protein containing the corresponding portion (MQASASDVVVVHGQRTP) of the peroxisomal thiolase appeared not to be localized to the mitochondria. These results show that the import signal of mitochondrial 3-oxoacyl-CoA thiolase originated from the NH2-terminal portion of the ancestral thiolase. The ancestral enzyme might have already possessed a mitochondrial import activity when mitochondria appeared first, or that it might have acquired the import activity during evolution by accumulation of point mutations in the NH2-terminal portion of the enzyme.

Molecular evolution from argininosuccinate lyase to delta-crystallin.

cDNA clones for rat argininosuccinate lyase, a urea cycle enzyme, were cloned and amino acid sequence of the enzyme was predicted. The rat enzyme is 54% identical with the yeast enzyme, which is involved in arginine biosynthesis, thereby indicating that this urea cycle enzyme evolved from the arginine biosynthetic enzyme. A striking similarity (64% identity) was found between amino acid sequences of rat argininosuccinate lyase and chicken delta-crystallin, a major structural protein of the eye lens. The gene for the rat argininosuccinate lyase was cloned and its structure was determined. This gene is a single-copy gene about 14 kilobases long and is split into 16 exons. A comparison with chicken delta-crystallin genes revealed that all introns interrupt the protein-coding regions at homologous positions. This close similarity in structural organization provides strong evidence for the view that the chicken delta 1- and delta 2-crystallin genes evolved by recruitment and duplication of the preexisting argininosuccinate lyase gene and that delta 2-crystallin is probably the direct homologue of argininosuccinate lyase.

Evolutionary aspects of urea cycle enzyme genes.

The functions and expression pattern of urea cycle enzymes have undergone considerable changes during the course of evolution. Sequence analyses shows that urea cycle enzymes from mammals are homologous to microbial enzymes of the arginine-metabolic pathway. Recently, an unexpected relationship was found between argininosuccinate lyase (EC 4.3.2.1), the fourth enzyme of the cycle, and delta-crystallin, a lens structural protein of birds and reptiles.

Molecular cloning and nucleotide sequence of rat brain argininosuccinate lyase cDNA with an extremely long 5'-untranslated sequence: evidence for the identity of the brain and liver enzymes.

Argininosuccinate lyase (EC 4.3.2.1) is an enzyme present in the brain of ureotelic animals. Using as a probe rat liver argininosuccinate lyase cDNA, already isolated and sequenced (Amaya, Y., Matsubasa, T., Takiguchi, M., Kobayashi, K., Saheki, T., Kawamoto, S. and Mori, M., J. Biochem., 103 (1988) 177-181), we screened a rat brain cDNA library constructed in the lambda gt11 expression vector and obtained a single cDNA clone. This cDNA clone contained an open reading frame encoding a polypeptide of 461 amino acid residues (predicted Mr = 51,390), a 5'-untranslated sequence of 967 bp and a 3'-untranslated sequence of 74 bp. The length of the 5'-non-coding region of the cDNA seems to be one of the longest among the cDNAs heretofore isolated. A comparison of the brain cDNA sequence (2424 bp) with the corresponding region of the liver cDNA (1574 bp) revealed differences in 5 nucleotides. The brain clone contained A----G and C----G base differences from the hepatic sequence, resulting in amino acid changes from Tyr and Arg in the liver clone, to Cys and Gly in the brain clone, respectively. The other 3 nucleotide differences are silent with respect to the amino acid sequence of the protein. Therefore, the amino acid sequence of the brain argininosuccinate lyase, as deduced from the nucleotide sequence of its cDNA clone, was identical with that of the liver protein, except for two amino acid residues. These minor changes may reflect a microheterogeneity of the argininosuccinate lyase gene. The brain and liver enzymes seem to be encoded by the same structural gene.

Structure of the rat argininosuccinate lyase gene: close similarity to chicken delta-crystallin genes.

Argininosuccinate lyase (EC 4.3.2.1) is an enzyme of arginine biosynthesis and is also involved in the urea cycle in the liver of ureotelic animals. A comparison of cDNA-derived amino acid sequences revealed that argininosuccinate lyase is highly homologous with chicken delta-crystallin, a major structural protein of the eye lens. The gene for the rat argininosuccinate lyase was cloned and its structure was determined. This gene is a single-copy gene about 14 kilobases long and is split into 16 exons. A comparison with chicken delta-crystallin genes revealed that all introns interrupt the protein-coding regions at homologous positions. This close similarity in structural organization provides strong evidence for the view of Piatigorsky et al. [Piatigorsky, J., O'Brien, W. E., Norman, B. L., Kalmuck, K., Wistow, G., Borras, T., Nickerson, J. M. & Wawrousek, E. F. (1988) Proc. Natl. Acad. Sci. USA 85, 3479-3483] that chicken delta 1- and delta 2-crystallin genes evolved by recruitment and duplication of the preexisting argininosuccinate lyase gene and that delta 2-crystallin is probably the direct homologue argininosuccinate lyase.

Reconstitution of mitochondrial protein transport with purified ornithine carbamoyltransferase precursor expressed in Escherichia coli.

Ornithine carbamoyltransferase (OTC; subunit, 36,000 Da) is initially synthesized as a precursor (pOTC) with a transient NH2-terminal presequence of 32 amino acid residues and imported posttranslationally into the mitochondrial matrix. The rat pOTC was synthesized in Escherichia coli using an expression vector containing a thermoinducible lambda pL promoter. The recombinant pOTC represented 5-10% of the total bacterial protein and was present in the precipitate of the disrupted bacteria. The precipitate was washed and pOTC was extracted with 8 M urea or 0.1% cetyltrimethylammonium bromide. The extracted pOTC was essentially homogeneous, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified pOTC was cleaved to the intermediate-sized product of 37,000 Da by a processing protease partially purified from the matrix fraction of rat liver mitochondria. The purified recombinant pOTC, but not the mature form of OTC synthesized in E. coli and purified, competed with the in vitro-synthesized, radiolabeled pOTC for uptake and processing by the isolated rat liver mitochondria. The radiolabeled and purified recombinant pOTC could be imported into the isolated mitochondria and processed to the mature form in an energy- and rabbit reticulocyte lysate-dependent manner. When the purified pOTC was subjected to sucrose gradient centrifugation, it sedimented as a large aggregate of greater than 60 S in the absence of reticulocyte lysate, whereas it sedimented as a complex of about 5 S in the presence of the lysate. These observations together with our previous results indicate that a protein factor(s) present in the lysate interacts with pOTC and holds it in an import-competent form.