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PubMed2006

A functional variant of the dopamine D3 receptor is associated with risk and age-at-onset of essential tremor.

Familial essential tremor (ET), the most common inherited movement disorder, is generally transmitted as an autosomal dominant trait. A genome-wide scan for ET revealed one major locus on chromosome 3q13. Here, we report that the Ser9Gly variant in the dopamine D(3) receptor gene (DRD3), localized on 3q13.3, is associated and cosegregates with familial ET in 23 out of 30 French families. Sequencing revealed no other nonsynonymous variants in the DRD3-coding sequence and in the first 871 bp of the 5' flanking region. Moreover, Gly-9 homozygous patients presented with more severe and/or earlier onset forms of the disease than heterozygotes. A replication study comparing 276 patients with ET and 184 normal controls confirmed the association of the Gly-9 variant with risk and age-at-onset of ET. In human embryonic kidney (HEK) 293-transfected cells, the Gly-9 variant did not differ from the Ser-9 variant with respect to glycosylation and to anterograde and retrograde trafficking, but dopamine had an affinity that was four to five times higher. With the Gly-9 variant, the dopamine-mediated cAMP response was increased, and the mitogen-associated protein kinase (MAPK) signal was prolonged, as compared with the Ser-9 variant. The gain-of-function produced by the Gly-9 variant may explain why drugs active against tremor in Parkinson's disease (PD) are usually not effective in the treatment of ET and suggests that DRD3 partial agonists or antagonists should be considered as novel therapeutic options for patients with ET.

Adolescent↗

PubMed1976

Molecular abnormality of human alpha1-antitrypsin variant (Pi-ZZ) associated with plasma activity deficiency.

A human alpha1-antitrypsin variant protein was purified to homogeneity from homozygous variant subjects (Pi-ZZ) who had a deficiency of plasma trypsin inhibitory capacity. Molecular weight, specific trypsin inhibitory capacity, and immunologic activity of the variant protein were identical to those of normal. Amino acids, N-acetylglucosamine, and hexose contents were closely similar in the normal and variant proteins, but the sialic acid content in the variant protein was significantly lower than normal. The structural difference between the normal and the variant alpha1-antitrypsin was elucidated by fingerprinting of their tryptic peptides. Two amino acid substitutions, i.e., glutamic acid in the normal protein to lysine in the variant protein, and glutamic acid in the normal protein to glutamine in the variant protein, were found.

Amino Acids↗

PubMed1982

Altered epidermal growth factor (EGF)-stimulated protein kinase activity in variant A431 cells with altered growth responses to EGF.

To determine the role of epidermal growth factor (EGF)-stimulated protein kinase in the biological effects caused by EGF, tyrosine-specific kinase activity has been quantitated in A431 human epidermoid carcinoma cells and six variant cell lines. Because EGF inhibited proliferation of A431 cells, variants resistant to this inhibition were selected by treatment with mutagen and maintenance for 1 month in 0.1 muM EGF. After cloning and growth for 6-20 generations without EGF, the resistance of the variants to the growth-inhibitory effect of EGF was confirmed. Whereas EGF increased cellular phosphotyrosine content approximately 10-fold in parental A431 cells, EGF caused smaller or undetectable increases in the six variant cell lines. Solubilized membranes from the six variants displayed diminished EGF-stimulated phosphorylation of the EGF receptor and of antibodies to p60(src) (the product of the Rous sarcoma virus transforming gene), which act as an exogenous substrate. The decrease in EGF-stimulated tyrosine-specific protein kinase activity varied from approximately 40% (clone 16) to approximately 8% (clone 18) of parental A431 activity. Phosphorylated EGF receptors from parental and variant cells migrated identically on sodium dodecyl sulfate/polyacrylamide gels. The number of EGF receptors in variant cells decreased in parallel with EGF-stimulated protein kinase activity, so that the specific activity of EGF-stimulated protein kinase per EGF receptor remained constant in the six variant cell lines with reductions in both activities to as low as 10%. These results suggest that this tyrosine-specific protein kinase activity mediates the growth-inhibitory effect of EGF on A431 cells and that both EGF binding and kinase activities reside in the same or tightly associated molecules.

Carcinoma, Squamous Cell↗

PubMed1993

Splice variants of the N-methyl-D-aspartate receptor NR1 identify domains involved in regulation by polyamines and protein kinase C.

The N-methyl-D-aspartate (NMDA) receptor NR1 gene encodes RNA that is alternatively spliced to generate at least seven variants. The variants arise from splicing in or out of three exons; one encodes a 21-amino acid insert in the N-terminal domain, and two encode adjacent sequences of 37 and 38 amino acids in the C-terminal domain. Splicing out of the second C-terminal exon deletes a stop codon and results in an additional open reading frame encoding an unrelated sequence of 22 amino acids before arriving at a second stop codon. We denote the NR1 variants by the presence or absence of the three alternatively spliced exons (from 5' to 3'); thus, NR1(111) has all three exons, NR1(000) has none, and NR1(100) has only the N-terminal exon. We report here electrophysiological characterization of six splice variants of the NR1 receptor expressed in Xenopus oocytes. NR1 receptors that lacked the N-terminal exon (NR1(000), NR1(010), and NR1(011)) exhibited a relatively high affinity for NMDA (EC50 approximately 13 microM) and marked potentiation by spermine. In contrast, those receptor variants with the N-terminal insert (NR1(100), NR1(101), and NR1(111)) showed a lower agonist affinity and little or no spermine potentiation at saturating glycine. All six variants showed spermine potentiation at low glycine and inhibition by spermine at more negative potentials. Variants differing only in the C-terminal domain differed little in agonist affinity and spermine potentiation. These findings indicate that the N-terminal insert either participates in agonist and polyamine binding domains or indirectly modifies their conformations. The splice variants differed in the extent to which they could be potentiated by activators of protein kinase C (PKC) from 3- to 20-fold. Presence of the N-terminal insert and absence of the C-terminal sequences increased potentiation by PKC. These findings identify the contributions of the separate polypeptide domains to modulation by polyamines and PKC and provide further support for the concept that subunit composition determines functional properties of NMDA receptors.

Alternative Splicing↗

PubMed1995

Four variant chicken erythroid AE1 anion exchangers. Role of the alternative N-terminal sequences in intracellular targeting in transfected human erythroleukemia cells.

Four variant AE1 anion exchangers with predicted molecular masses of approximately 99, approximately 102, approximately 104, and approximately 108 kDa are expressed in chicken erythroid cells. These variant polypeptides differ in sequence only at the N terminus of their cytoplasmic domains. Molecular analyses have shown that transcripts derived from both of the erythroid-specific promoters, P1 and P2, encode all four of these AE1 anion exchanger variants. However, quantitative RNase protection analyses have shown that the transcripts derived from the P1 promoter are much more prevalent than those derived from the P2 promoter. Reverse transcriptase polymerase chain reaction studies have indicated that the extensive diversity in the transcripts derived from the AE1 gene occurs both in primitive and definitive lineage erythroid cells. Transient transfection analyses using human erythroleukemia cells have investigated the functional significance of the alternative sequences at the N terminus of these variant exchangers. These studies have shown that the erythroid AE1 variants are sorted to different membrane compartments in these cells. The approximately 99- and approximately 102-kDa variants are primarily sorted to the plasma membrane, whereas the approximately 108-kDa variant is retained in a perinuclear compartment. These results suggest that the alternative N-terminal cytoplasmic sequences of these polypeptides may serve as signals to direct these variant transporters to different membrane compartments within cells.

Amino Acid Sequence↗

PubMed1995

Different antifolate-resistant L1210 cell variants with either increased or decreased folylpolyglutamate synthetase gene expression at the level of mRNA transcription.

L1210 cell variants selected in the presence of the lipophilic dihydrofolate reductase inhibitor, metoprine, expressed increased levels of one-carbon, reduced folate transport inward (Sirotnak, F. M., Moccio, D. M., and Yang, C.-H. (1984) J. Biol. Chem. 259, 13139-13144). Growth of one of these variants (L1210/R69), with metoprine in the presence of decreasing concentrations of 1,L5-CHO-folateH4 (natural diastereoisomer of 5-formyl-tetrahydrofolate), resulted in the selection of other variants (L1210/R82, R83, and R84) with further reduction in one-carbon, reduced folate transport and in two cases (L1210/R83 and R84) with 3-8-fold increased folylpolyglutamate synthetase (FPGS) activity and folate compound polyglutamate formation in situ. Metoprine resistance was further increased, and the requirement for exogenous folate during growth was decreased as well in these variants. The increase in FPGS activity observed in L1210/R83 and R84 was characterized by 3- and 8-fold increases in value for Vmax with no change in Km and the same increase in a 60-61-kDa protein as shown by immunoblotting. Northern blotting revealed the same increases in these two variants in the level of a 2.3-kilobase FPGS mRNA when compared with control, while Southern blotting of genomic DNA did not reveal any increase in FPGS gene-copy number or restriction polymorphisms. Also, no difference in stability of FPGS mRNA was found between parental and variant cells. In contrast, nuclear run-on assays revealed differences among these cell types in the rate of FPGS mRNA transcription that correlated with increased FPGS activity, protein, and mRNA level in the variants. Similar studies with a transport-defective, methotrexate-resistant L1210 cell variant (L1210/R25) documented a 2-3-fold decrease in FPGS activity, protein, and mRNA levels that was accounted for by a decrease in FPGS mRNA transcription. These results provide the first examples of constitutively altered transcriptional regulation of FPGS activity associated with acquired resistance to antifolates.

Amino Acid Sequence↗

PubMed1998

Improved potency of hyperactive and actin-resistant human DNase I variants for treatment of cystic fibrosis and systemic lupus erythematosus.

The ability of recombinant human DNase I (DNase I) to degrade DNA to lower molecular weight fragments is the basis for its therapeutic use in cystic fibrosis (CF) patients and its potential use as a treatment for systemic lupus erythematosus (SLE). To increase the potency of human DNase I, we have generated and characterized three classes of mutants: (a) hyperactive variants, which have from one to six additional positively charged residues (+1 to +6) and digest DNA much more efficiently relative to wild type, (b) actin-resistant variants, which are no longer inhibited by G-actin, a potent inhibitor of DNase I, and (c) combination variants that are both hyperactive and actin-resistant. For DNA scission in CF sputum where the DNA concentration and length are large, we measured a approximately 20-fold increase in potency relative to wild type for the +3 hyperactive variant Q9R/E13R/N74K or the actin-resistant variant A114F; the hyperactive and actin-resistant combination variant was approximately 100-fold more potent than wild type DNase I. For digesting lower concentrations of DNA complexed to anti-DNA antibodies in human serum, we found a maximal enhancement of approximately 400-fold over wild type for the +2 variant E13R/N74K. The +3 enzymes have approximately 4000-fold enhancement for degrading moderate levels of exogenous DNA spiked into human serum, whereas the +6 enzyme has approximately 30,000-fold increased activity for digesting the extremely low levels of endogenous DNA found in serum. The actin resistance property of the combination mutants further enhances the degree of potency in human serum. Thus, the human DNase I variants we have engineered for improved biochemical and pharmacodynamic properties have greater therapeutic potential for treatment of both CF and SLE.

Actins↗

PubMed2002

Alternative splice variants of doublecortin-like kinase are differentially expressed and have different kinase activities.

Alternative splicing of mRNA transcripts expands the range of protein products from a single gene locus. Several splice variants of DCLK (doublecortin-like kinase) have previously been reported. Here, we report the genomic organization underlying the splice variants of DCLK and examine the expression profile of two splice variants affecting the kinase domain of DCLK and CPG16 (candidate plasticity gene 16), one containing an Arg-rich domain and the other affecting the C terminus of the protein. These splice alternatives were differentially expressed in embryonic and adult brain. Both splice variants disrupted DCLK PEST domains; however, all splice variants remained sensitive to proteolysis by calpain. The adult-specific C-terminal splice variant of DCLK had reduced autophosphorylation activity, but similar kinase activity for myelin basic protein relative to the embryonic splice variant. The splice variant adding an Arg-rich domain gained an autophosphorylation site at Ser-382. Although this protein isoform was expressed mainly in the adult brain, the phosphorylated form was strongly enriched in embryonic brain and adult olfactory bulb, suggesting a possible role in migrating neurons.

Alternative Splicing↗

PubMed2002

Tat HIV-1 primary and tertiary structures critical to immune response against non-homologous variants.

Clinical studies show that in the absence of anti-retroviral therapy an immune response against the human immunodeficiency virus type 1 (HIV-1), transacting transcriptional activator (Tat) protein correlates with long term non-progression. The purpose of this study is to try to understand what can trigger an effective immune response against Tat. We used five Tat variants from HIV strains identified in different parts of the world and showed that mutations of as much as 38% exist without any change in activity. Rabbit sera were raised against Tat variants identified in rapid-progressor patients (Tat HXB2, a European variant and Tat Eli, an African variant) and a long term non-progressor patient (Tat Oyi, an inactive African variant). Enzyme-linked immunosorbent assay (ELISA) results showed that anti-Tat Oyi serum had the highest antibody titer and was the only one to have a broad antibody response against heterologous Tat variants. Surprisingly, Tat HXB2 was better recognized by anti-Tat Oyi serum compared with anti-Tat HXB2 serum. Western blots showed that non-homologous Tat variants were recognized by antibodies directed against conformational epitopes. This study suggests that the primary and tertiary structures of the Tat variant from the long term non-progressor patient are critical to the induction of a broad and effective antibody response against Tat.

AIDS Vaccines↗

PubMed2004

Influence of the unusual covalent adduct on the kinetics and formation of radical intermediates in synechocystis catalase peroxidase: a stopped-flow and EPR characterization of the MET275, TYR249, and ARG439 variants.

Catalase-peroxidases (KatGs) are heme peroxidases with a catalatic activity comparable to monofunctional catalases. They contain an unusual covalent distal side adduct with the side chains of Trp(122), Tyr(249), and Met(275) (Synechocysis KatG numbering). The known crystal structures suggest that Tyr(249) and Met(275) could be within hydrogen-bonding distance to Arg(439). To investigate the role of this peculiar adduct, the variants Y249F, M275I, R439A, and R439N were investigated by electronic absorption, steady-state and transient-state kinetic techniques and EPR spectroscopy combined with deuterium labeling. Exchange of these conserved residues exhibited dramatic consequences on the bifunctional activity of this peroxidase. The turnover numbers of catalase activity of M275I, Y249F, R439A, and R439N are 0.6, 0.17, 4.9, and 3.14% of wild-type activity, respectively. By contrast, the peroxidase activity was unaffected or even enhanced, in particular for the M275I variant. As shown by mass spectrometry and EPR spectra, the KatG typical adduct is intact in both Arg(439) variants, as is the case of the wild-type enzyme, whereas in the M275I variant the covalent link exists only between Tyr(249) and Trp(122). In the Y249F variant, the link is absent. EPR studies showed that the radical species formed upon reaction of the Y249F and R439A/N variants with peroxoacetic acid are the oxoferryl-porphyrin radical, the tryptophanyl and the tyrosyl radicals, as in the wild-type enzyme. The dramatic loss in catalase activity of the Y249F variant allowed the comparison of the radical species formed with hydrogen peroxide and peroxoacetic acid. The EPR data strongly suggest that the sequence of intermediates formed in the absence of a one electron donor substrate, is por(.-)(+) --> Trp(.-) (or Trp(.-)(+)) --> Tyr(.-). The M275I variant did not form the Trp(.-) species because of the dramatic changes on the heme distal side, most probably induced by the repositioning of the remaining Trp(122)-Tyr(249) adduct. The results are discussed with respect to the bifunctional activity of catalase-peroxidases.

Bacterial Proteins↗

PubMed2005

The unique NH2-terminally deleted (DeltaN) residues, the PXXP motif, and the PPXY motif are required for the transcriptional activity of the DeltaN variant of p63.

p63, a member of the p53 family of transcription factors, is known to be involved in epithelial development. However, its role in tumorigenesis is unclear. Contributing to this uncertainty, the TP63 locus can express multiple gene products from two different promoters. Utilization of the upstream promoter results in expression of the TAp63 variant with an activation domain similar to p53. In contrast, the NH2-terminally deleted (DeltaN) p63 variant, transcribed from a cryptic promoter in intron 3, lacks such an activation domain. Thus, the TAp63 and DeltaNp63 variants possess a wide ranging ability to up-regulate p53 target genes. Consequentially, the disparity in transactivation potential between p63 variants has given rise to the hypothesis that the DeltaNp63 variant can serve as oncoprotein by opposing the activity of the TAp63 variant and p53. However, recent studies have revealed a transcriptional activity for DeltaNp63. This study was undertaken to address the transcriptional activity of the DeltaNp63 variant. Here, we showed that all NH2-terminally deleted p63 isoforms retain a potential in transactivation and growth suppression. Interestingly, DeltaNp63beta possesses a remarkable ability to suppress cell proliferation and transactivate target genes, which is consistently higher than that seen with DeltaNp63alpha. In contrast, DeltaNp63gamma has a weak or undetectable activity dependent upon the cell lines used. We also demonstrate that an intact DNA-binding domain is required for DeltaNp63 function. In addition, we found that the novel activation domain for the DeltaNp63 variant is composed of the 14 unique DeltaN residues along with the adjacent region, including a PXXP motif. Finally, we demonstrated that a PPXY motif shared by DeltaNp63alpha and DeltaNp63beta is required for optimal transactivation of target gene promoters, suggesting that the PPXY motif is requisite for DeltaNp63 function.

Amino Acid Sequence↗

PubMed2006

Contribution of CARD15 variants in determining susceptibility to Crohn's disease in Sweden.

OBJECTIVE: Crohn's disease (CD) is a chronic inflammatory bowel disorder caused by environmental and genetic factors. Mutations in the CARD15 gene have been associated with CD. No previous case-control CARD15 study has been performed in the Swedish population. MATERIAL AND METHODS: The study comprised of 321 individuals: 178 with CD and 143 healthy controls (HCs), all from Stockholm County. All were genotyped for the three main CD-associated CARD15 variants (R702W, G908R and 1007fs) and phenotypic associations were investigated. RESULTS: The allele frequencies of the R702W variant (4.5% CD versus 0.7% HC, p=0.008, OR = 6.8) and the G908R variant (2.0% CD versus 0% HC, p=0.045) were more common in CD patients than in controls. No significant difference in1007fs variant allele frequency was found between CD patients and controls (2.0% CD versus 1.7% HC, p = 0.8, OR = 1.1). Carriage of CARD15 variants was more common in the CD patients than in controls (15.2% CD versus 4.2% HC, p = 0.001, OR = 4.1, population attributable risk (PAR) = 11.4%). Genotype-phenotype analysis demonstrated that CARD15 variants were associated with ileal disease (p=0.0006, OR = 9.3, CI = 2.2-34) and protective for colonic CD (p = 0.01, OR = 0.18). An association between CARD15 variants and ileal CD (p=0.004, OR = 6.6) was confirmed by multivariate analyses. CONCLUSIONS: The CARD15 variants R702W and G908R, but not 1007fs, are associated with susceptibility to CD in Stockholm County. Genotype-phenotype analysis shows an association with ileal CD. The contribution of these CARD15 mutations in Swedish CD patients overall is low in relation to studies elsewhere in Central Europe and North America, but is consistent with emerging data from elsewhere in Scandinavia and in Northern Europe.

Alleles↗

PubMed2023

Phylogeny and evolution of SARS-CoV-2 during Delta and Omicron variant waves in India.

SARS-CoV-2 evolution has continued to generate variants, responsible for new pandemic waves locally and globally. Varying disease presentation and severity has been ascribed to inherent variant characteristics and vaccine immunity. This study analyzed genomic data from 305 whole genome sequences from SARS-CoV-2 patients before and through the third wave in India. Delta variant was reported in patients without comorbidity (97%), while Omicron BA.2 was reported in patients with comorbidity (77%). Tissue adaptation studies brought forth higher propensity of Omicron variants to bronchial tissue than lung, contrary to observation in Delta variants from Delhi. Study of codon usage pattern distinguished the prevalent variants, clustering them separately, Omicron BA.2 isolated in February grouped away from December strains, and all BA.2 after December acquired a new mutation S959P in ORF1b (44.3% of BA.2 in the study) indicating ongoing evolution. Loss of critical spike mutations in Omicron BA.2 and gain of immune evasion mutations including G142D, reported in Delta but absent in BA.1, and S371F instead of S371L in BA.1 could explain very brief period of BA.1 in December 2021, followed by complete replacement by BA.2. Higher propensity of Omicron variants to bronchial tissue, probably ensured increased transmission while Omicron BA.2 became the prevalent variant possibly due to evolutionary trade-off. Virus evolution continues to shape the epidemic and its culmination.Communicated by Ramaswamy H. Sarma.

SARS-CoV-2↗

PubMed2002

Frequency and significance of antibodies to soluble liver antigen/liver pancreas in variant autoimmune hepatitis.

BACKGROUND: Antibodies to soluble liver antigen/liver pancreas are highly specific markers of autoimmune hepatitis. AIMS: Determine the frequency and clinical significance of these antibodies in the variant syndromes. METHODS: Antibodies to soluble liver antigen/liver pancreas were determined in 28 patients with variant forms, including 10 with cryptogenic chronic hepatitis and 18 with cholestatic variants. One hundred and seventy-two patients with classical autoimmune hepatitis were similarly tested. RESULTS: Seven of the 28 patients with variant forms had the antibodies, and this frequency was not statistically different than that in classical disease (25 vs. 12%, p = 0.08). Antibodies were most common in patients with cryptogenic chronic hepatitis (40%). Seropositive patients were indistinguishable from seronegative patients with variant forms, and they responded as well to corticosteroid therapy as patients with autoimmune hepatitis. Relapse after corticosteroid withdrawal invariably occurred in the seropositive patients whether with variant or classical disease, and HLA DR3 was more common in the seropositive patients with variant forms than in normal subjects (60 vs. 15%, p = 0.03). CONCLUSIONS: Antibodies to soluble liver antigen/liver pancreas occur commonly in the variant forms of autoimmune hepatitis and identify patients that closely resemble classical disease. Seropositivity is associated with relapse after corticosteroid withdrawal and HLA DR3. The antibodies may be surrogate markers of a genetic propensity to relapse that is independent of clinical phenotype.

Autoantibodies↗

PubMed1992

The dermal-epidermal junction of human skin contains a novel laminin variant.

We report the identification of a novel laminin variant that appears to be unique to a subset of epithelial basement membranes. The variant contains two chains electrophoretically and immunologically identical to the B1 and B2 chains. Epitopes contained in the laminin A chain are absent from the molecule, and a 190-kD chain substitutes for the A chain. V8 protease analysis and Western blotting studies indicate that the variant 190-kD chain shows structural and immunological similarity to the 200-kD chain of kalinin. Rotary shadowing analysis indicates that the 190-kD chain contributes a large globular structure to the variant long arm, but lacks the short arm contributed to laminin by the A chain. The variant is produced by cultured skin explants, human keratinocytes and a squamous cell carcinoma line, and is present in human amniotic fluid. Polyclonal antibodies raised to kalinin, a recently characterized novel component of anchoring filaments, and mAb BM165 which recognizes a subunit of kalinin (Rousselle et al., 1991) cross react with the variant under nonreducing conditions. Immunohistological surveys of human tissues using the crossreacting antikalinin antiserum indicate that the distribution of this laminin variant is at least restricted to anchoring filament containing basement membranes. We propose the name K-laminin for this variant.

Basement Membrane↗

PubMed1993

Biochemical characterization and tissue distribution of the A and B variants of the integrin alpha 6 subunit.

Two cytoplasmic variants of the alpha 6 integrin, alpha 6A and alpha 6B, have been identified previously (Hogervorst, F., I. Kuikman, A. G. van Kessel, and A. Sonnenberg. 1991. Eur. J. Biochem. 199:425-433; Cooper, H. M., R. N. Tamura, and V. Quaranta. 1991. J. Cell Biol. 115:843-850). Using synthetic peptides, containing sequences of their cytoplasmic domains, we have produced mAbs specific for either of the variants. These antibodies reacted with a variety of different epithelial tissues. In some tissues (e.g., salivary gland) both variants could be detected while in others only one of the variants was found (e.g., alpha 6A in epidermis and alpha 6B in kidney). Among nonepithelial cells and tissues, perineural fibroblasts and Schwann cells in peripheral nerves and platelets reacted with anti-alpha 6A, while microvascular endothelia reacted with both anti-alpha 6A and anti-alpha 6B. From our immunohistochemical results there is not evidence that combination with beta 1 or beta 4 is restricted to one of the two variants of alpha 6. This was confirmed by immunoprecipitation studies which showed that both beta 1 and beta 4 were coprecipitated by both anti-alpha 6A or anti-alpha 6B antibodies from cells. Also, the distribution of alpha 6A and alpha 6B subunits associated with beta 1 on cells attached to laminin was similar: both were found in focal contacts colocalizing with vinculin. In contrast, the alpha 6A subunit, associated with beta 4 in cultures of a squamous cell carcinoma cell line, was found to codistribute with bullous pemphigoid antigen 230 in hemidesmosomal-like structures. The alpha 6A and alpha 6B variants, immunoprecipitated from various cell lines, exhibited slightly different electrophoretic mobilities. Analysis of the antigens under reducing conditions showed that the mobility of the light chains, but not of the heavy chains, is different. In addition, in some cells the light chains of alpha 6A and alpha 6B, each are of two different sizes. Treatment with N-glycanase showed that these two light chain variants of alpha 6A and alpha 6B are not due to differences in N-linked glycosylation, and may therefore represent alternative proteolytic products of the alpha 6 precursor. We further demonstrate that alpha 6A, but not alpha 6B, is a major target for PMA-induced phosphorylation. Phosphorylated alpha 6A contained phosphoserine and a small amount of phosphotyrosine. There are also two variants of the integrin alpha 3 subunit with different cytoplasmic domains, but in the cell lines examined only alpha 3A could be demonstrated by RT-PCR.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗

PubMed1995

Large and small splice variants of collagen XII: differential expression and ligand binding.

Collagen XII has a short collagenous tail and a very large, three-armed NC3 domains consisting primarily of fibronectin type III repeats. Differential splicing within this domain gives rise to a large (320 kD) and a small (220 kD) subunit; the large but not the small can carry glycosaminoglycan. To investigate whether collagen XII variants have distinct expression patterns and functions, we generated antibody and cDNA probes specific for the alternatively spliced domain. We report here that the large variant has a more restricted expression in embryonic tissue than the small. For example, whereas the small variant is widespread in the dermis, the large is limited to the base of feather buds. Distinct proportions of mRNA for the two variants were detected depending on the tissue. Monoclonal antibodies allowed us to separate collagen XII variants, and to show that homo- and heterotrimers exist. Collagen XII variants differ in ligand binding. Small subunits interact weakly with heparin via their COOH-terminal domain. Large subunits have additional, stronger heparin-binding site(s) in their NH2-terminal extra domain. In vivo, both large and small collagen XII are associated with interstitial collagen. Here we show biochemically and ultrastructurally that collagen XII can be incorporated into collagen I fibrils when it is present during, but not after, fibril formation. Removal of the collagenous domain of collagen XII reduces its coprecipitation with collagen I. Our results indicate that collagen XII is specifically associated with fibrillar collagen, and that the large variant has binding sites for extracellular ligands not present in the small variant.

Alternative Splicing↗

PubMed2001

Niemann-Pick C variant detection by altered sphingolipid trafficking and correlation with mutations within a specific domain of NPC1.

Niemann-Pick disease type C (NPC) is a fatal, autosomal recessive lipidosis characterized by lysosomal accumulation of unesterified cholesterol and multiple neurological symptoms, such as vertical supranuclear ophthalmoplegia, progressive ataxia, and dementia. More than 90% of cases of NPC are due to a defect in Niemann-Pick C1 (NPC1), a late endosomal, integral membrane protein that plays a role in cholesterol transport or homeostasis. Biochemical diagnosis of NPC has relied on the use of patient skin fibroblasts in an assay to demonstrate delayed low-density lipoprotein (LDL)-derived cholesterol esterification and a cytological technique-filipin staining-to demonstrate the intracellular accumulation of cholesterol. A small percentage of patients, referred to as "NPC variants," present with clinical symptoms of NPC but show near-normal results of these biochemical tests, making laboratory confirmation of NPC disease problematic. Here, we demonstrate that NPC-variant fibroblast samples can be detected as sphingolipid storage disease cells, using a fluorescent sphingolipid analog, BODIPY-lactosylceramide. This lipid accumulated in endosomes/lysosomes in variant cells preincubated with LDL cholesterol but targeted to the Golgi complex in normal cells under these conditions. The reproducibility of this technique was validated in a blinded study. In addition, we performed mutation analysis of the NPC1 gene in NPC variant and "classical" NPC cell samples and found a high incidence of specific mutations within the cysteine-rich region of NPC1 in variants. We also found that 5 of the 12 variant cell samples had no apparent defect in NPC1 but were otherwise indistinguishable from other variant cells. This is a surprising result, since, in general, approximately 90% of patients with NPC possess defects in NPC1. Our findings should be useful for the detection of NPC variants and also may provide significant new insight regarding NPC1 genotype/phenotype correlations.

Alleles↗