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PubMed1987

Nucleotide sequence of medium-chain acyl-CoA dehydrogenase mRNA and its expression in enzyme-deficient human tissue.

Medium-chain acyl-CoA dehydrogenase (MCAD; acyl-CoA: (acceptor) 2,3-oxidoreductase, EC 1.3.99.3) is one of three similar enzymes that catalyze the initial step of fatty acid beta-oxidation. Definition of the primary structure of MCAD and the tissue distribution of its mRNA is of biochemical and clinical importance because of the recent recognition of inherited MCAD deficiency in humans. The MCAD mRNA nucleotide sequence was determined from two overlapping cDNA clones isolated from human liver and placental cDNA libraries, respectively. The MCAD mRNA includes a 1263-base-pair coding region and a 738-base-pair 3'-nontranslated region. A partial amino acid sequence (137 residues) determined on peptides derived from MCAD purified from porcine liver confirmed the identity of the cDNA clone. Comparison of the amino acid sequence predicted from the human MCAD cDNA with the partial protein sequence of the porcine MCAD revealed a high degree (88%) of interspecies sequence identity. RNA blot analysis shows that MCAD mRNA is expressed in a variety of rat (2.2 kilobases) and human (2.4 kilobases) tissues. Blot hybridization of RNA prepared from cultured skin fibroblasts from a patient with MCAD deficiency disclosed that mRNA was present and of similar size to MCAD mRNA derived from control fibroblasts. The isolation and characterization of MCAD cDNA is an important step in the definition of the defect underlying MCAD deficiency and in understanding its metabolic consequences.

Acyl-CoA Dehydrogenase↗

PubMed1996

ECA39, a conserved gene regulated by c-Myc in mice, is involved in G1/S cell cycle regulation in yeast.

The c-myc oncogene has been shown to play a role in cell proliferation and apoptosis. The realization that myc oncogenes may control the level of expression of other genes has opened the field to search for genetic targets for Myc regulation. Recently, using a subtraction/coexpression strategy, a murine genetic target for Myc regulation, called EC439, was isolated. To further characterize the ECA39 gene, we set out to determine the evolutionary conservation of its regulatory and coding sequences. We describe the human, nematode, and budding yeast homologs of the mouse ECA39 gene. Identities between the mouse ECA39 protein and the human, nematode, or yeast proteins are 79%, 52%, and 49%, respectively. Interestingly, the recognition site for Myc binding, located 3' to the start site of transcription in the mouse gene, is also conserved in the human homolog. This regulatory element is missing in the ECA39 homologs from nematode or yeast, which also lack the regulator c-myc. To understand the function of ECA39, we deleted the gene from the yeast genome. Disruption of ECA39 which is a recessive mutation that leads to a marked alteration in the cell cycle. Mutant haploids and homozygous diploids have a faster growth rate than isogenic wild-type strains. Fluorescence-activated cell sorter analyses indicate that the mutation shortens the G1 stage in the cell cycle. Moreover, mutant strains show higher rates of UV-induced mutations. The results suggest that the product of ECA39 is involved in the regulation of G1 to S transition.

Amino Acid Sequence↗

PubMed1999

Modeling and predicting transcriptional units of Escherichia coli genes using hidden Markov models.

MOTIVATION: The hidden Markov model (HMM) is a valuable technique for gene-finding, especially because its flexibility enables the inclusion of various sequence features. Recent programs for bacterial gene-finding include the information of ribosomal binding site (RBS) to improve the recognition accuracy of the start codon, using this feature. We report here our attempt to extend the model into the total transcriptional unit, enabling the prediction of operon structures. RESULTS: First, we improved the prediction accuracy of coding sequences (CDSs) by employing the models of 'typical', 'atypical' and 'negative (false-positive)' classes as well as the models of RBS and its downstream spacer. The sensitivity of exactly predicting the 204 experimentally confirmed CDSs reached 90.2% in an objective test. Based on the prediction result of CDSs, the positions of the promoters and terminators were predicted. Our model could exactly recognize 60% of 390 known transcriptional units. Thus, the accuracy and significance of this prediction problem is far from trivial. We would like to propose this problem as an open theme in bioinformatics because the ongoing or planned post-sequencing projects will produce much data for future improvements.

Algorithms↗

PubMed1989

cDNA molecular cloning of Geotrichum candidum lipase.

The cDNA clone of Geotrichum candidum (Geo.) lipase was isolated from the Geo. cDNA library by colony hybridization using 32P-labeled oligonucleotides corresponding to a partial amino acid sequence of this enzyme. The nucleotide sequence of the cDNA determined by the dideoxy chain terminating method included some partial amino acid sequences determined by Edman degradation, and the overall amino acid composition deduced from the cDNA coincided with that from amino acid analysis of this protein. The cloned cDNA coded a protein of 554 amino acids and a hydrophobic signal sequence of 19 amino acids. Geo. lipase contained the -Gly-X-Ser-X-Gly- sequence which is believed to form part of the interfacial lipid recognition site.

Amino Acid Sequence↗

PubMed1995

Molecular evolution of mitochondrial 12S RNA and cytochrome b sequences in the pantherine lineage of Felidae.

DNA sequence comparisons of two mitochondrial DNA genes were used to infer phylogenetic relationships among 17 Felidae species, notably 15 in the previously described pantherine lineage. The polymerase chain reaction (PCR) was used to generate sequences of 358 base pairs of the mitochondrial 12S RNA gene and 289 base pairs of the cytochrome b protein coding gene. DNA sequences were compared within and between 17 felid and five nonfelid carnivore species. Evolutionary trees were constructed using phenetic, cladistic, and maximum likelihood algorithms. The combined results suggested several phylogenetic relationships including (1) the recognition of a recently evolved monophyletic genus Panthera consisting of Panthera leo, P. pardus, P. onca, P. uncia, P. tigris, and Neofelis nebulosa; (2) the recent common ancestry of Acinonyx jubatus, the African cheetah, and Puma concolor, the American puma; and (3) two golden cat species, Profelis temmincki and Profelis aurata, are not sister species, and the latter is strongly associated with Caracal caracal. These data add to the growing database of vertebrate mtDNA sequences and, given the relatively recent divergence among the felids represented here (1-10 Myr), allow 12S and cytochrome b sequence evolution to be addressed over a time scale different from those addressed in most work on vertebrate mtDNA.

Acinonyx↗

PubMed1998

The production of English inflectional morphology, speech production and listening performance in children with cochlear implants.

OBJECTIVE: To compare how children who use either cochlear implants (CIs) or hearing aids (HAs) express English inflectional morphemes during conversation, i.e., with voice, with sign, or with both. A secondary objective was to investigate the relationship between morpheme use in pediatric CI users and their speech perception skills, length of experience with the device, and accuracy of phoneme production. DESIGN: Group 1 consisted of 25 children who used CIs, and Group 2 consisted of 13 children who used HAs. All children were prelingually deafened and all used simultaneous communication. A 12 minute spontaneous conversation was elicited, transcribed and coded. Between group comparisons were performed to evaluate differences in modality and number of morphemes used. Additionally, use of morpheme endings was related to length of CI experience, accuracy of phoneme production, and closed-set speech recognition performance. RESULTS: Children who had CI experience produced significantly more English inflected morphemes than children in the HA group. CI participants also expressed the inflected endings by using voice-only mode 91% of the time, whereas HA participants used voice-only mode 1% of the time. In the CI group, a strong relationship was found between number of morpheme endings used and speech recognition scores, length of CI experience and accuracy of phoneme production. The results of this study indicate that input from the CI facilitates children's ability to perceive and comprehend bound morphemes.

Adolescent↗

PubMed1998

Suppression of human anti-porcine T-cell immune responses by major histocompatibility complex class II transactivator constructs lacking the amino terminal domain.

BACKGROUND: The class II transactivator (CIITA) is a bi- or multifunctional domain protein that acts as a transcriptional activator and plays a critical role in the expression of MHC class II genes. We have previously demonstrated that a mutated form of the human CIITA gene, coding for a protein lacking the amino terminal 151 amino acids, acts as a potent dominant-negative suppressor of HLA class II expression. Porcine MHC class II antigens are potent stimulators of direct T-cell recognition by human CD4+ T cells and are, therefore, likely to play an important role in the rejection responses to transgenic pig donors in clinical xenotransplantation. We were, therefore, interested in examining mutated CIITA constructs for their effect on porcine MHC class II expression. METHODS: Stable transfectants of the porcine vascular endothelial cell line PIEC with mutated CIITA constructs were tested for SLA-DR and SLA-DQ induction by recombinant porcine interferon-gamma. Transient transfectants of the porcine B-cell line L23 with the mutated CIITA constructs were tested for the suppression of constitutive SLA-DR and SLA-DQ expression. T-cell proliferation studies were performed using highly purified human CD4+ T cells. RESULTS: In preliminary studies, we demonstrated that transfection of the PIEC line with full-length human CIITA constructs resulted in strong expression of SLA-DR and SLA-DQ antigens, thus establishing the cross-species effectiveness of human CIITA in the pig. The mutated human CIITA constructs were, therefore, tested in the pig. PIEC clones stably transfected with one of these constructs showed up to 99% suppression of SLA-DR and SLA-DQ antigen induction and marked suppression of SLA-DRA mRNA induction. Moreover, transient transfection of the porcine B-cell line L23 showed up to 90% suppression of constitutive SLA-DR and SLA-DQ antigen expression in 5-8 days. In functional studies, interferon-gamma-stimulated PIEC clones transfected with this mutated CIITA construct failed to stimulate purified human CD4+ T lymphocytes. CONCLUSION: Mutated human CIITA constructs are potent suppressors of porcine MHC class II expression.

Amino Acid Sequence↗

PubMed1990

A monoclonal antibody specific to zeatin o-glycosyltransferases of phaseolus.

Zeatin O-xylosyltransferase and zeatin O-glucosyltransferase occur in immature embryos of Phaseolus vulgaris and P. lunatus, respectively. Purified preparations of the xylosyltransferase were used as antigen to elicit the formation of antibodies in mice. Hybridoma clones were produced by fusion of mouse spleen cells with myeloma cell line Fox-NY. A clone secreting monoclonal antibody (MAb), XZT-1, capable of immunoprecipitating both enzymes was obtained. The MAb detected a unique protein band from crude embryo extracts of each species with the correct molecular mass (50 kilodaltons) and relative charge (R(F) = 0.5 and 0.3) of the respective enzymes. Competition experiments with substrates indicated that the glycosyl dinucleotide binding sites of the enzymes are probably not involved in MAb-enzyme recognition. Western blotting of samples from vegetative tissues of P. vulgaris detected a low level of O-glucosyltransferase but not O-xylosyltransferase, in leaves. These findings suggest the occurrence of two genes in P. vulgaris coding for O-glycosylation enzymes with tissue-specific expression. The MAb will be used to screen expression libraries and to obtain pure enzymes for amino acid sequencing and for the production of additional MAbs.

Journal Article↗

PubMed1996

Enhanced degradation of EGF receptors by a sorting nexin, SNX1.

The vectorial movement of proteins requires specific recognition by components of the vesicular trafficking machinery. A protein, sorting nexin-1 (SNX1), was identified in a human cell line that bound to a region of the epidermal growth factor receptor (EGFR) containing the lysosomal targeting code. SNX1 contains a region of homology to a yeast vacuolar sorting protein, and overexpression of SNX1 decreased the amount of EGFR on the cell surface as a result of enhanced rates of constitutive and ligand-induced degradation. Thus, SNX1 is likely to play a role in sorting EGFR to lysosomes.

Amino Acid Sequence↗

PubMed1993

Isolation of a glutamate synthase (GOGAT)-negative, pleiotropically N utilization-defective mutant of Azospirillum brasilense: cloning and partial characterization of GOGAT structural gene.

An Azospirillum brasilense mutant (N12) pleiotropically defective in the assimilation of nitrogenous compounds (Asm-) was isolated and found lacking in the glutamate synthase (GOGAT-). The glt (GOGAT) locus of A. brasilense was identified by isolating a broad-host-range pLAFR1 cosmid clone from a gene library of the bacterium that rectified Asm- and GOGAT- defects (full recovery of activities of the nitrogenase, the assimilatory nitrate and nitrite reductases, and the glutamate synthase). A 7.5-kb EcoRI fragment of the cosmid clone that also complemented N12 was partially sequenced to identify the open reading frame for the alpha-subunit of GOGAT. The amino acid sequences deduced from the partial nucleotide sequences of the glt locus of A. brasilense showed considerable homology with that of the alpha-subunit of GOGAT coded by the gltB gene of Escherichia coli. The genetic lesion of N12 was found within the gltB gene of A. brasilense. The gltB promoter of A. brasilense showed the presence of a consensus sigma-70-like recognition site (as in E. coli) in addition to potential NtrA-RNA polymerase, IHF, and NifA binding sites.

Amino Acid Sequence↗

PubMed1993

A transthyretin variant (alanine 71) associated with familial amyloidotic polyneuropathy in a French family.

A transthyretin (TTR) mutation is described in a 44 year old French woman from Caen who presented at the age of 40 with neuropathy in all four extremities, diarrhoea, and orthostatic hypotension. Her father died with a similar syndrome including vitreous opacities. A nerve biopsy from the proband showed amyloid deposits which stained with anti-transthyretin. Direct genomic DNA sequencing of TTR exon 3 showed both thymine and cytosine in the position corresponding to the second base of codon 71. This codes for a variant alanine (GCG) as well as the normal valine (GTG), indicating that the proband is heterozygous for the substitution. Since this substitution does not result in the creation or abolition of a restriction endonuclease recognition site, a new technique (PCR-IMRA) was used to create an RFLP. Using a 24 bp nucleotide mutagenesis primer in the PCR reaction, a new NspBII site is created on amplification of the variant allele. With this method a 170 bp TTR exon 3 PCR product was generated for both the normal and the variant allele. On digestion of the PCR product with NspBII, DNA from a heterozygous subject showed both the 170 bp undigested product from the normal allele and a 146 bp digestion product from the variant allele. By PCR-IMRA, two of five children of the proband were positive for the variant allele. This non-radioactive technique gives a rapid method for testing subjects at risk for this mutation.

Adult↗

PubMed1995

Molecular mechanisms of antiasthma therapy.

Recently there has been a much greater understanding of the molecular mechanisms involved in the actions of antiasthma therapy. beta 2-agonists are the most effective bronchodilators and act predominantly on airway smooth muscle. Recent evidence suggests that beta 2-receptors in airway smooth muscle are coupled directly to maxi-K channels and may thereby bronchodilate without an increase in cyclic AMP. The issue of beta-receptor tolerance has been reawakened by the recognition that the protective effects of beta 2-agonists against bronchoconstrictor stimuli may become tolerant. Inhaled glucocorticoids are the mainstay of treatment in patients with chronic asthma. They suppress asthmatic inflammation predominantly by reducing transcription of genes coding for inflammatory mediators (particularly cytokines) and enzymes (inducible NO synthase, inducible cyclo-oxygenase). The inhibition of gene transcription is mediated predominantly by inhibition of transcription factors, such as activator protein-1 (AP-1) and nuclear factor-kappa B (NF-kappa B). There may be an abnormal activation of AP-1 in steroid-resistant asthma, and high concentrations of beta 2-agonists may induce a secondary resistance by an interaction between the transcription factor CREB and the glucocorticoid receptor. Theophylline may have immunomodulatory effects that are more important than its bronchodilator action. Some effects of theophylline are mediated via inhibition of phosphodiesterases and several PDE IV inhibitors are currently undergoing evaluation in asthma.

Adrenergic beta-Agonists↗

PubMed1999

Structure and chromosomal assignment of the human lectin-like oxidized low-density-lipoprotein receptor-1 (LOX-1) gene.

We have reported the cDNA cloning of a modified low-density-lipoprotein (LDL) receptor, designated lectin-like oxidized LDL receptor-1 (LOX-1), which is postulated to be involved in endothelial dysfunction and the pathogenesis of atherosclerosis. Here, we determined the organization of the human LOX-1 gene, including the 5'-regulatory region. The 5'-regulatory region contained several potential cis-regulatory elements, such as GATA-2 binding element, c-ets-1 binding element, 12-O-tetradecanoylphorbol 13-acetate-responsive element and shear-stress-responsive elements, which may mediate the endothelium-specific and inducible expression of LOX-1. The major transcription-initiation site was found to be located 29 nucleotides downstream of the TATA box and 61 nucleotides upstream from the translation-initiation codon. The minor initiation site was found to be 5 bp downstream from the major site. Most of the promoter activity of the LOX-1 gene was ascribed to the region (-150 to -90) containing the GC and CAAT boxes. The coding sequence was divided into 6 exons by 5 introns. The first 3 exons corresponded to the different functional domains of the protein (cytoplasmic, transmembrane and neck domains), and the residual 3 exons encoded the carbohydrate-recognition domain similar to the case of other C-type lectin genes. The LOX-1 gene was a single-copy gene and assigned to the p12.3-p13.2 region of chromosome 12. Since the locus for a familial hypertension has been mapped to the overlapping region, LOX-1 might be the gene responsible for the hypertension.

Base Sequence↗

PubMed2005

[Isolation and functional analysis of tobacco MARs].

Two new MAR segments (M14 and M17) were cloned from tobacco genome. Both of the sequences contained several typical consensus sequences of MARs, which were different from the original MAR sequence, such as 90%AT-box, A-box, T-box, the base unpairing regions (BUR), autonomously replicating sequences (ARS), the consensus sequence for topoisomerase II, MAR recognition sequence (MRS), origin of replication (ORI), curved DNA motifs and ATATTT et al. To investigate the effects of these two sequences on gene expression in transgenic plants, 3 plant expression vectors were constructed with uidA gene coding beta-glucuronidase (GUS) which were flanked on one side and on both sides by the MARs we obtained. These plant expression vectors with one or two MARs were transformed into tobaccos via Agrobacterium-mediated transformation method, with the plant expression vector pCAMBIA2301 without MAR and wild type tobacco as controls. GUS histochemical staining results showed that the uidA gene expressed stably in transgenic tobaccos. Quantitative detection of GUS activity showed that the MARs could increase GUS expression levels in vivo in contrast to the controls, wherever they were flanked on one side or both sides of uidA gene. The vector ligated with MARs in the same direction on both sides of uidA could increase the GUS expression level much better than both vectors which just ligated with single MARs on one side. The former one increased the average GUS activity for 3.14 folds, but 1.56 and 2.43 folds for the latter two vectors with single MARs respectively contrasting to the pCAMBIA2301 control. But the expression differences among individual transformants were still obvious. Therefore, it was concluded that the DNA sequences we obtained in this experiment were two novel MARs and could enhance gene expression in vivo. In the meanwhile, although the numbers of the MARs typical motifs in M14 were more than in M17, especially the 90% AT box which had been considered to be the highest correlative motif with binding strength in vitro, the enhancement of gene expression was lower yet, which implied no correlation between improvement of gene expression and binding strength between MARs and nuclear matrix in vitro.

Cloning, Molecular↗

PubMed1993

Selective in vitro expansion of HLA class I-restricted HIV-1 Gag-specific CD8+ T cells: cytotoxic T-lymphocyte epitopes and precursor frequencies.

OBJECTIVE: To identify HIV-1 Gag cytotoxic T-lymphocyte (CTL) epitopes and HLA restriction of their recognition, and to define precursor frequencies of HIV-1 Gag-specific CTL in the blood of seropositive individuals. METHODS: B-lymphoblastoid cell lines (B-LCL) infected with recombinant vaccinia viruses (rVV) containing a gene coding for HIV-1 Gag (rVV-Gag) were fixed with paraformaldehyde (PFA) and used as antigen-presenting cells (APC) to stimulate peripheral blood mononuclear cells (PBMC) from asymptomatic HIV-seropositive individuals. Specific CTL activity was determined in 51Cr-release assays using B-LCL as targets after infection with rVV-Gag or after pulsing with partially overlapping peptides spanning the Gag sequence. RESULTS: In vitro stimulation resulted in an increased number of CD8+ T cells and CD45R0+ and HLA-DR+ cells. Gag-specific cytotoxicity, mediated predominantly by HLA class I-restricted CD8+ CTL, was observed in all seven individuals studied. Multiple HLA-restricted CTL epitopes were identified with a single culture from one of the individuals. Gag-expressing APC were successfully used as stimulator cells in limiting dilution analysis to determine CTL precursor (CTLp) frequencies. CONCLUSION: PFA-fixed rVV-Gag-infected autologous B-LCL can be used as stimulator cells in bulk PBMC cultures to identify CTL epitopes and to determine CTLp frequencies. This method will facilitate the analysis of HIV-1-specific CTL responses in HIV-infected and vaccinated individuals.

Antigen-Presenting Cells↗

PubMed2005

Different strategies for carboxyl-terminal domain (CTD) recognition by serine 5-specific CTD phosphatases.

The phosphorylated carboxyl-terminal domain (CTD) of RNA polymerase II, consisting of ((1)YSPTSPS(7))(n) heptad repeats, encodes information about the state of the transcriptional apparatus that can be conveyed to factors that regulate mRNA synthesis and processing. Here we describe how the CTD code is read by two classes of protein phosphatases, plant CPLs and yeast Ssu72, that specifically dephosphorylate Ser(5) in vitro. The CPLs and Ssu72 recognize entirely different positional cues in the CTD primary structure. Whereas the CPLs rely on Tyr(1) and Pro(3) located on the upstream side of the Ser(5)-PO(4) target site, Ssu72 recognizes Thr(4) and Pro(6) flanking the target Ser(5)-PO(4) plus the downstream Tyr(1) residue of the adjacent heptad. We surmise that the reading of the CTD code does not obey uniform rules with respect to the location and phasing of specificity determinants. Thus, CTD code, like the CTD structure, is plastic.

Amino Acid Sequence↗

PubMed1987

Specific residues within an amino-terminal domain of 35 residues of interferon alpha are responsible for recognition of the human interferon alpha cell receptor and for triggering biological effects.

Bovine interferon alpha C (IFN-alpha C) manifest at least 10(5)-fold lower antiviral activity on human cells than on bovine cells (Velan, B., Cohen, S., Grosfeld, H., Leitner, M., and Shafferman, A. (1985) J. Biol. Chem. 260, 5498-5504). By oligonucleotide site-directed mutagenesis within the coding region for the NH2-terminal 44-residue domain of BoIFN-alpha C, we replaced up to 18 residues by the corresponding HuIFN-alpha J1 residues. (HuIFN-alpha J1 is less than 60% homologous in sequence to BoIFN-alpha C.) The nine different bovine-human-IFN alpha hybrids obtained were compared to BoIFN-alpha C and HuIFN-alpha J1 with respect to their potential to induce an antiviral state, synthesis of 2-5A-synthetase, and their specific binding to human and bovine cells. Relative to BoIFN-alpha C, a gradual increase in biological activities (antiviral or 2-5A-synthetase) of approximately 10-, 10(2)-, 10(3)-, and approximately 10(4)-fold is obtained, depending on the number and positions of the residues substituted. A direct correlation exists between biological response and ability of IFN alpha to bind specifically to human cells. A BoIFN alpha molecule mutated in the 10-44 NH2-terminal domain was obtained which is 15, 8, and 35% as active as HuIFN-alpha J1 on human cells in specific binding, induction of antiviral, and 2-5A-synthetase activities, respectively. We concluded that at least 5 of the 12 residues at positions 10; 21, 22, 24; 27; 31, 34, 35, 37, 40; 42, 43 in the 10-44 NH2-terminal domain are critical for recognition of the human IFN-alpha cell receptor and for biological activity. These residues are found among 10 strictly conserved residues in all reported mammalian IFN alpha S, and they act in a cooperative manner to induce a biological response in human cells. The gap between the extent of improvement in binding capacity of the BoIFN alpha mutants on human cells and the corresponding biological response suggests that the primary signal of binding to the cell receptor is amplified within the cell. On bovine cells, HuIFN-alpha J1 and BoIFN-alpha C also compete for the same receptor, and it seems that at least part of the 10-44 NH2-terminal domain on IFN alpha is also involved in interaction with the bovine IFN alpha cell receptor.

2',5'-Oligoadenylate Synthetase↗

PubMed1999

Adhesive domains in the collagen III fragment alpha1(III)CB4 that support alpha2beta1- and von Willebrand factor-mediated platelet adhesion under flow conditions.

Seven overlapping peptides derived from the bovine alpha1(III)CB4 fragment of collagen III support static platelet adhesion, and an integrin alpha2beta1-recognition site has been assigned within this fragment to residues 522-528 of the collagen alpha1(III) chain; (25). In this study we found that two of the peptides, CB4(III)-6 and -7, were able to support platelet adhesion under flow conditions, whereas the other peptides showed either very little (CB4(III)-1 and -4) or no platelet adhesion at all (CB4(III)-2, -3 and -5). Using the recombinant leech anti-platelet protein (rLAPP), known to prevent both alpha2beta1 integrin- and von Willebrand factor (vWF)-binding to collagen, we observed almost complete inhibition of platelet adhesion to peptides CB4(III)-6 and -7. In solid-phase binding assays rLAPP bound to CB4(III)-6 and -7 and to CB4(III)-6/7, containing the peptide 6/7 overlap sequence, and not to any other peptide. Our results suggest that the overlap sequence GPP*GPRGGAGPP*GPEGGK (single-letter amino acid code, P* = hydroxyproline), corresponding to residues 523-540 of the alpha1(III) collagen chain, contains a binding site for rLAPP. Monoclonal antibodies (MoAbs) directed against the alpha2 subunit of integrin alpha2beta1 inhibited platelet adhesion to both CB4(III)-6 and -7 by about 50%, showing that the alpha2beta1-recognition site in this locality in alpha1(III)CB4 detected under static conditions is of sufficient affinity to withstand shear forces. Solid-phase binding studies indicated that vWF binds to CB4(III)-7 and to a lesser extent to CB4(III)-4. Furthermore, rLAPP competed with vWF in binding to CB4(III)-7. Our results indicate that residues 541-558 of the alpha1(III)-chain may contain one of the critical vWF-binding sites involved in the initial phase of platelet adhesion to collagen III. MoAbs against vWF (A1 and A3 domain) and glycoprotein (GP)Ib confirmed that vWF is involved in adhesion to CB4(III)-7 and showed that vWF is also involved in adhesion to CB4(III)-6 despite the absence of direct binding of vWF to the peptide. The existence of alpha2beta1-, vWF- and rLAPP-binding sites all in close proximity in alpha1(III)CB4 testifies to the importance of this locus in collagen III for its platelet reactivity.

Amino Acid Sequence↗