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1.
Antimicrob Agents Chemother ; 64(5)2020 Apr 21.
Artigo em Inglês | MEDLINE | ID: mdl-32152074

RESUMO

In the past few decades, enterovirus A71 (EVA71) has caused devastating outbreaks in the Asia-Pacific region, resulting in serious sequelae in infected young children. No preventive or therapeutic interventions are currently available for curing EVA71 infection, highlighting a great unmet medical need for this disease. Here, we showed that one novel single-domain antibody (sdAb), F1, isolated from an immunized llama, could alleviate EVA71 infection both in vitro and in vivo We also confirmed that the sdAb clone F1 recognizes EVA71 through a novel conformational epitope comprising the highly conserved region of VP3 capsid protein by using competitive-binding and overlapping-peptide enzyme-linked immunosorbent assays (ELISAs). Because of the virion's icosahedral structure, we reasoned that adjacent epitopes must be clustered within molecular ranges that may be simultaneously bound by an engineered antibody with multiple valency. Therefore, two single-domain binding modules (F1) were fused to generate an sdAb-in-tandem design so that the capture of viral antigens could be further increased by valency effects. We showed that the tetravalent construct F1×F1-hFc, containing two sdAb-in-tandem on a fragment crystallizable (Fc) scaffold, exhibits more potent neutralization activity against EVA71 than does the bivalent sdAb F1-hFc by at least 5.8-fold. We also demonstrated that, using a human scavenger receptor class B member 2 (hSCARB2) transgenic mouse model, a half dose of the F1×F1-hFc provided better protection against EVA71 infection than did the F1-hFc. Thus, our study furnishes important insights into multivalent sdAb engineering against viral infection and provides a novel strategic deployment approach for preparedness of emerging infectious diseases such as EVA71.

2.
Nucleic Acids Res ; 46(15): e93, 2018 09 06.
Artigo em Inglês | MEDLINE | ID: mdl-29905834

RESUMO

Baculovirus (BV) holds promise as a vector for anticancer gene delivery to combat the most common liver cancer-hepatocellular carcinoma (HCC). However, in vivo BV administration inevitably results in BV entry into non-HCC normal cells, leaky anticancer gene expression and possible toxicity. To improve the safety, we employed synthetic biology to engineer BV for transgene expression regulation. We first uncovered that miR-196a and miR-126 are exclusively expressed in HCC and normal cells, respectively, which allowed us to engineer a sensor based on distinct miRNA expression signature. We next assembled a synthetic switch by coupling the miRNA sensor and RNA binding protein L7Ae for translational repression, and incorporated the entire device into a single BV. The recombinant BV efficiently entered HCC and normal cells and enabled cis-acting transgene expression control, by turning OFF transgene expression in normal cells while switching ON transgene expression in HCC cells. Using pro-apoptotic hBax as the transgene, the switch-based BV selectively killed HCC cells in separate culture and mixed culture of HCC and normal cells. These data demonstrate the potential of synthetic switch-based BV to distinguish HCC and non-HCC normal cells for selective transgene expression control and killing of HCC cells.


Assuntos
Baculoviridae/genética , Carcinoma Hepatocelular/terapia , Regulação Neoplásica da Expressão Gênica , Neoplasias Hepáticas/terapia , MicroRNAs/genética , Transgenes/genética , Animais , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Sobrevivência Celular/genética , Vetores Genéticos/genética , Células HEK293 , Células Hep G2 , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , MicroRNAs/metabolismo , Reprodutibilidade dos Testes , Células Sf9 , Spodoptera , Biologia Sintética/métodos
3.
Biochem Biophys Rep ; 9: 51-60, 2017 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-28955989

RESUMO

QBEND/10 is a mouse immunoglobulin lambda-chain monoclonal antibody with strict specificity against human hematopoietic progenitor cell antigen CD34. Our in vitro study showed that QBEND/10 impairs the tube formation of human umbilical vein endothelial cells (HUVECs), suggesting that the antibody may be of potential benefit in blocking tumor angiogenesis. We provided a de novo protein sequencing method through tandem mass spectrometry to identify the amino acid sequences in the variable heavy and light chains of QBEND/10. To reduce immunogenicity for clinical applications, QBEND/10 was further humanized using the resurfacing approach. We demonstrate that the de novo sequenced and humanized QBEND/10 retains the biological functions of the parental mouse counterpart, including the binding kinetics to CD34 and blockage of the tube formation of the HUVECs.

4.
ACS Appl Mater Interfaces ; 8(22): 13714-23, 2016 Jun 08.
Artigo em Inglês | MEDLINE | ID: mdl-27198164

RESUMO

Fluorescent silicon quantum dots (SiQDs) have shown a great potential as antiphotobleaching, nontoxic and biodegradable labels for various in vitro and in vivo applications. However, fabricating SiQDs with high water-solubility and high photoluminescence quantum yield (PLQY) remains a challenge. Furthermore, for targeted imaging, their surface chemistry has to be capable of conjugating to antibodies, as well as sufficiently antifouling. Herein, antibody-conjugated SiQD nanoparticles (SiQD-NPs) with antifouling coatings composed of bovine serum albumin (BSA) and polyethylene glycol (PEG) are demonstrated for immunostaining on live cancer cells. The monodisperse SiQD-NPs of diameter about 130 nm are synthesized by a novel top-down method, including electrochemical etching, photochemical hydrosilylation, high energy ball milling, and "selective-etching" in HNO3 and HF. Subsequently, the BSA and PEG are covalently grafted on to the SiQD-NP surface through presynthesized chemical linkers, resulting in a stable, hydrophilic, and antifouling organic capping layer with isothiocyanates as the terminal functional groups for facile conjugation to the antibodies. The in vitro cell viability assay reveals that the BSA-coated SiQD-NPs had exceptional biocompatibility, with minimal cytotoxicity at concentration up to 1600 µg mL(-1). Under 365 nm excitation, the SiQD-NP colloid emits bright reddish photoluminescence with PLQY = 45-55% in organic solvent and 5-10% in aqueous buffer. Finally, through confocal fluorescent imaging and flow cytometry analysis, the anti-HER2 conjugated SiQD-NPs show obvious specific binding to the HER2-overexpressing SKOV3 cells and negligible nonspecific binding to the HER2-nonexpressing CHO cells. Under similar experimental conditions, the immunofluorescence results obtained with the SiQD-NPs are comparable to those using conventional fluorescein isothiocyanate (FITC).


Assuntos
Nanopartículas , Pontos Quânticos , Coloração e Rotulagem/métodos , Animais , Sobrevivência Celular/efeitos dos fármacos , Cricetinae , Cricetulus , Nanopartículas/toxicidade , Polietilenoglicóis/química , Soroalbumina Bovina/química , Silício
5.
Theranostics ; 6(1): 118-30, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-26722378

RESUMO

The overexpression of HER2/neu and EGFR receptors plays important roles in tumorigenesis and tumor progression. Targeting these two receptors simultaneously can have a more widespread application in early diagnosis of cancers. In this study, a new multifunctional nanoparticles (MnMEIO-CyTE777-(Bis)-mPEG NPs) comprising a manganese-doped iron oxide nanoparticle core (MnMEIO), a silane-amino functionalized poly(ethylene glycol) copolymer shell, a near infrared fluorescence dye (CyTE777), and a covalently conjugated anti-HER2/neu and anti-EGFR receptors bispecific antibody (Bis) were successfully developed. In vitro T2-weighted MR imaging studies in SKBR-3 and A431 tumor cells incubated with MnMEIO-CyTE777-(Bis)-mPEG NPs showed - 94.8 ± 3.8 and - 84.1 ± 2.8% negative contrast enhancement, respectively. Pharmacokinetics study showed that MnMEIO-CyTE777-(Bis)-mPEG NPs were eliminated from serum with the half-life of 21.3 mins. In vivo MR imaging showed that MnMEIO-CyTE777-(Bis)-mPEG NPs could specifically and effectively target to HER2/neu- and EGFR-expressing tumors in mice; the relative contrast enhancements were 11.8 (at 2 hrs post-injection) and 61.5 (at 24 hrs post-injection) fold higher in SKBR-3 tumors as compared to Colo-205 tumors. T2-weighted MR and optical imaging studies revealed that the new contrast agent (MnMEIO-CyTE777-(Bis)-mPEG NPs) could specifically and effectively target to HER2/neu- and/or EGFR-expressing tumors. Our results demonstrate that MnMEIO-CyTE777-(Bis)-mPEG NPs are able to recognize the tumors expressing both HER2/neu and/or EGFR, and may provide a novel molecular imaging tool for early diagnosis of cancers expressing HER2/neu and/or EGFR.


Assuntos
Anticorpos Biespecíficos/farmacocinética , Receptores ErbB/análise , Compostos Férricos/farmacocinética , Imagem por Ressonância Magnética/métodos , Manganês/farmacocinética , Neoplasias/diagnóstico , Receptor ErbB-2/análise , Animais , Magnetismo , Camundongos , Neoplasias/patologia , Coloração e Rotulagem/métodos
6.
Int J Nanomedicine ; 10: 3663-85, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26056447

RESUMO

We have developed a theranostic nanoparticle, ie, cet-PEG-dexSPIONs, by conjugation of the anti-epidermal growth factor receptor (EGFR) monoclonal antibody, cetuximab, to dextran-coated superparamagnetic iron oxide nanoparticles (SPIONs) via periodate oxidation. Approximately 31 antibody molecules were conjugated to each nanoparticle. Cet-PEG-dexSPIONs specifically bind to EGFR-expressing tumor cells and enhance image contrast on magnetic resonance imaging. Cet-PEG-dexSPION-treated A431 cells showed significant inhibition of epidermal growth factor-induced EGFR phosphorylation and enhancement of EGFR internalization and degradation. In addition, a significant increase in apoptosis was detected in EGFR-overexpressing cell lines, A431 and 32D/EGFR, after 24 hours of incubation at 37°C with cet-PEG-dexSPIONs compared with cetuximab alone. The antibody-dependent cell-mediated cytotoxicity of cetuximab was observed in cet-PEG-dexSPIONs. The results demonstrated that cet-PEG-dexSPIONs retained the therapeutic effect of cetuximab in addition to having the ability to target and image EGFR-expressing tumors. Cet-PEG-dexSPIONs represent a promising targeted magnetic probe for early detection and treatment of EGFR-expressing tumor cells.


Assuntos
Cetuximab/química , Cetuximab/farmacologia , Dextranos/química , Receptores ErbB/metabolismo , Imagem por Ressonância Magnética/métodos , Nanopartículas de Magnetita/química , Anticorpos Monoclonais/química , Anticorpos Monoclonais/farmacologia , Antineoplásicos/química , Antineoplásicos/farmacologia , Linhagem Celular Tumoral/efeitos dos fármacos , Dextranos/farmacologia , Receptores ErbB/imunologia , Humanos , Microscopia Eletrônica de Transmissão , Terapia de Alvo Molecular/métodos , Nanomedicina Teranóstica/métodos
7.
Anal Chem ; 87(1): 545-53, 2015 Jan 06.
Artigo em Inglês | MEDLINE | ID: mdl-25427836

RESUMO

Prostate specific antigen (PSA) is a widely used serum marker for prostate cancer (PCa), but has limited specificity for distinguishing early PCa from benign prostatic hyperplasia (BPH). Recently, proPSAs, comprised of native proPSA, as well as truncated proPSA forms, [-2] proPSA, [-5] proPSA, and [-7] proPSA, have been shown to be better diagnostic targets than PSA for PCa. Stable isotope labeling-multiple reaction monitoring mass spectrometry (SIL/MRM-MS) has been frequently used to measure low-abundance biomarkers in tissues and biofluids, owing to its high sensitivity and specificity, simplicity, and multiplexing capability. In this study, we have developed and optimized a strategy using immunoprecipitation in conjunction with SIL/MRM-MS assay which is capable of sensitive and accurate quantification of proPSA in serum. Since serum and plasma are by far the most complex biological fluids, the immunoprecipitation workflow was optimized to achieve sufficient sensitivity, efficiencies of protein purification with immunoaffinity depletion were determined. The developed strategy can detect proPSA and PSA with a limit of detection (LOD) and limit of quantitation (LOQ) at nanogram per milliliter levels, corresponding to a concentration 6 orders-of-magnitude lower than the most abundant serum proteins. Furthermore, the simultaneous measurement of multiple biomarkers, including the mature and precursor forms of PSA, can be achieved in a single multiplexed analysis using LC/MRM-MS. The strategy demonstrated here provides an attractive alternative to ELISAs or RIAs for the reliably measurement of proPSA to improve the specificity of PCa diagnosis.


Assuntos
Proteínas Sanguíneas/análise , Imunoprecipitação/métodos , Espectrometria de Massas/métodos , Fragmentos de Peptídeos/análise , Antígeno Prostático Específico/sangue , Neoplasias da Próstata/diagnóstico , Bioensaio , Cromatografia Líquida , Feminino , Voluntários Saudáveis , Humanos , Marcação por Isótopo , Masculino , Neoplasias da Próstata/sangue , Isoformas de Proteínas , Radioimunoensaio
8.
Anal Chem ; 84(11): 4900-6, 2012 Jun 05.
Artigo em Inglês | MEDLINE | ID: mdl-22519857

RESUMO

An automatic method for disulfide bond assignment using dimethyl labeling and computational screening of a(1) ions with customized software, RADAR, is developed. By utilization of the enhanced a(1) ions generated from labeled peptides, the N-terminal amino acids from disulfide-linked peptides can be determined. In this study, we applied this method for structural characterization of recombinant monoclonal antibodies, an important group of therapeutic proteins. In addition to a(1) ion screening and molecular weight match, new RADAR is capable of confirming the matched peptide pairs by further comparing the collision-induced dissociation (CID) fragment ions. With the N-terminal amino acid identities as a threshold, the identification of disulfide-linked peptide pairs can be achieved rapidly at a higher confidence level. Unlike most current approaches, prior knowledge of disulfide linkages or a high-end mass spectrometer is not required, and tedious work or deliberate interpretation can be avoided in this study. Our approach makes it possible to analyze unknown disulfide bonds of protein pharmaceuticals as well as their degraded forms without further protein separation. It can be used as a convenient quality examination tool during biopharmaceutical development and manufacturing processes.


Assuntos
Anticorpos Monoclonais/química , Dissulfetos/química , Proteínas Recombinantes/química , Software , Sequência de Aminoácidos , Química Farmacêutica , Humanos , Íons , Dados de Sequência Molecular , Espectrometria de Massas por Ionização por Electrospray , Coloração e Rotulagem , Espectrometria de Massas em Tandem
9.
PLoS One ; 6(1): e16373, 2011 Jan 31.
Artigo em Inglês | MEDLINE | ID: mdl-21305012

RESUMO

A murine monoclonal antibody, m357, showing the highly neutralizing activities for human tumor necrosis factor (TNF-α) was chosen to be humanized by a variable domain resurfacing approach. The non-conserved surface residues in the framework regions of both the heavy and light chain variable regions were identified via a molecular modeling of m357 built by computer-assisted homology modeling. By replacing these critical surface residues with the human counterparts, a humanized version, h357, was generated. The humanized h357 IgG(1) was then stably expressed in a mammalian cell line and the purified antibody maintained the high antigen binding affinity as compared with the parental m357 based on a soluble TNF-α neutralization bioassay. Furthermore, h357 IgG(1) possesses the ability to mediate antibody-dependent cell-mediated cytotoxicity and complement dependent cytotoxicity upon binding to cells bearing the transmembrane form of TNF-α. In a mouse model of collagen antibody-induced arthritis, h357 IgG significantly inhibited disease progression by intra-peritoneal injection of 50 µg/mouse once-daily for 9 consecutive days. These results provided a basis for the development of h357 IgG as therapeutic use.


Assuntos
Anticorpos Monoclonais/genética , Engenharia de Proteínas/métodos , Fator de Necrose Tumoral alfa/imunologia , Animais , Anticorpos Monoclonais/imunologia , Afinidade de Anticorpos , Artrite Experimental/tratamento farmacológico , Humanos , Imunoglobulina G , Camundongos , Modelos Moleculares , Mutagênese Sítio-Dirigida , Resultado do Tratamento
10.
Genes Chromosomes Cancer ; 48(12): 1057-68, 2009 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-19760608

RESUMO

Investigating aberrant DNA methylation in the cancer genome may identify genes that play an important role in tumor progression. In this study, we combined differential methylation hybridization and a CpG microarray platform to characterize methylation profiles and identify novel candidate genes associated with hepatocellular carcinoma (HCC). The genomic DNA of 21 paired adjacent normal and HCC samples was used, and results were analyzed by hierarchical clustering. Twenty-seven hypermethylated candidates and 38 hypomethylated candidates were obtained. Six candidate genes from the hypermethylated group were validated by combined bisulfite restriction analysis; two genes, human kallikrein 10 gene (KLK10) and oxoglutarate (alpha-ketoglutarate) receptor 1 gene (OXGR1), were further analyzed by bisulfite sequencing. The DNA hypermethylation status of KLK10 and OXGR1 were subsequently examined in HCC cell lines and clinical samples using methylation-specific PCR. In 49 HCC samples, 46 (94%) showed that at least one of these two genes was highly methylated. Moreover, KLK10 and OXGR1 mRNA levels were inversely correlated (r = -0.435 and -0.497, P < 0.05) with DNA methylation as examined in paired adjacent normal and tumor samples. Statistical analyses further indicated that KLK10 hypermethylation was significantly associated with cirrhosis (P = 0.042) and HCV infection (P = 0.017) as well as inversely associated with HBV infection (P = 0.023). Furthermore, restoration of KLK10 and OXGR1 expression reduced the ability of anchorage-independent growth, and sensitized HCC cells to doxorubicin- or 5-fluorouracil-induced cytotoxicity. Our results suggest that the hypermethylated KLK10 and OXGR1 are frequent in HCC and may be useful as markers for clinical application.


Assuntos
Carcinoma Hepatocelular/genética , Metilação de DNA , Regulação Neoplásica da Expressão Gênica , Calicreínas/genética , Neoplasias Hepáticas/genética , Receptores Acoplados a Proteínas-G/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Western Blotting , Carcinoma Hepatocelular/patologia , Ensaio de Unidades Formadoras de Colônias , Ilhas de CpG , Feminino , Humanos , Neoplasias Hepáticas/patologia , Masculino , Pessoa de Meia-Idade , Análise de Sequência com Séries de Oligonucleotídeos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células Tumorais Cultivadas
11.
FASEB J ; 22(11): 3795-804, 2008 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-18635738

RESUMO

A class of multivalent protein binders was designed to overcome the limitations of low-affinity therapeutic antibodies. These binders, termed "collabodies," use a triplex-forming collagen-like peptide to drive the trimerization of a heterologous target-binding domain. Different forms of collabody, consisting of the human single-chain variable fragment (scFv) fused to either the N or C terminus of the collagen-like peptide scaffold (Gly-Pro-Pro)(10), were stably expressed as soluble secretory proteins in mammalian cells. The collabody consisting of scFv fused to the N terminus of collagen scaffold is present as a homotrimer, whereas it exhibited a mixture of trimer and interchain disulfide-bonded hexamer when cysteine residues were introduced and flanked the scaffold. The collagenous motif in collabody is prolyl-hydroxylated, with remarkable thermal and serum stabilities. The collabody erb_scFv-Col bound to the extracellular domain of epidermal growth factor receptor with a binding strength approximately 20- and 1000-fold stronger than the bivalent and monovalent counterparts, respectively. The trimeric collagen scaffold does not compromise the functionality of the binding moieties of parental immunoglobulin G (IgG); therefore, it could be applied to fuse other protein molecules to acquire significantly improved targeting-binding strengths.


Assuntos
Colágeno/química , Receptores ErbB/química , Região Variável de Imunoglobulina/química , Peptídeos/química , Animais , Linhagem Celular Tumoral , Colágeno/genética , Colágeno/metabolismo , Receptores ErbB/genética , Receptores ErbB/metabolismo , Humanos , Região Variável de Imunoglobulina/genética , Região Variável de Imunoglobulina/metabolismo , Camundongos , Peptídeos/genética , Peptídeos/metabolismo , Ligação Proteica/genética , Estrutura Quaternária de Proteína/genética , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo
12.
Biochim Biophys Acta ; 1784(2): 312-8, 2008 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-17980171

RESUMO

The CDA14, a 45kD protein, is currently annotated as PTX1-like protein or ERGIC 32. Over expressing CDA14 can slow PC11 cell proliferation rate. In HepG2 cells, it had been demonstrated that CDA14 is involved in protein transportation. The knowledge about the protein is very limited and not clarified. The CDA14 and its homologous proteins form a family and are restricted to eukaryotes. In the family, there are no homologous sequences with resolved three-dimensional structure and their functions are difficult to predict. Transcriptional expression of CDA14 in three hepatoma cell lines, Hu7, HCC and HepG2, was lower than normal liver tissue and liver carcinoma tissue. In this study, functional proteomic techniques were utilized in searching the interacting counterpart of CDA14. Several proteins involved in protein translation and folding were selectively precipitated with CDA14 and identified mass spectrometry. Interaction of CDA14 and elongation factor 1alpha was confirmed by Western blotting and confocal microscopy. Elongation factor 1alpha is a multiple function protein and involved in several biological mechanisms, including protein synthesis, cell proliferation, apoptosis and tumorigensis. Over-expression of CDA14 down regulated the proliferation of HepG2 cells. These results suggest that CDA14 participated in the elongation factor 1alpha regulated mechanisms.


Assuntos
Fator 1 de Elongação de Peptídeos/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Humanos , Ligação Proteica , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/isolamento & purificação
13.
Biochem Biophys Res Commun ; 336(2): 375-85, 2005 Oct 21.
Artigo em Inglês | MEDLINE | ID: mdl-16115607

RESUMO

We established stably transfected insect cell lines containing cDNAs encoding the alpha and beta subunits of human prolyl 4-hydroxylase in both Trichoplusia ni and Drosophila melanogaster S2 cells. The expression level and enzymatic activity of recombinant prolyl 4-hydroxylase produced in the Drosophila expression system were significantly higher than those produced in the T. ni system. We further characterized the involvement of prolyl 4-hydroxylase in the assembly of the three alpha chains to form trimeric type XXI minicollagen, which comprises the intact C-terminal non-collagenous (NC1) and collagenous domain (COL1), in the Drosophila system. When minicollagen XXI was stably expressed in Drosophila S2 cells alone, negligible amounts of interchain disulfide-bonded trimers were detected in the culture media. However, minicollagen XXI was secreted as disulfide-bonded homotrimers by coexpression with prolyl 4-hydroxylase in the stably transfected Drosophila S2 cells. Minicollagen XXI coexpressed with prolyl 4-hydroxylase contained sufficient amounts of hydroxyproline to form thermal stable pepsin-resistant triple helices consisting of both interchain and non-interchain disulfide-bonded trimers. These results demonstrate that a sufficient amount of active prolyl 4-hydroxylase is required for the assembly of type XXI collagen triple helices in Drosophila cells and the trimeric assembly is governed by the C-terminal collagenous domain.


Assuntos
Drosophila melanogaster/metabolismo , Colágenos Fibrilares/química , Colágenos Fibrilares/metabolismo , Mariposas/metabolismo , Pró-Colágeno-Prolina Dioxigenase/química , Pró-Colágeno-Prolina Dioxigenase/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Linhagem Celular , Dimerização , Drosophila melanogaster/genética , Colágenos Fibrilares/análise , Colágenos Fibrilares/genética , Dados de Sequência Molecular , Mariposas/genética , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Pró-Colágeno-Prolina Dioxigenase/análise , Pró-Colágeno-Prolina Dioxigenase/genética , Ligação Proteica , Proteínas Recombinantes/análise , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Transfecção/métodos
14.
Genomics ; 79(3): 395-401, 2002 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-11863369

RESUMO

We cloned a 4.1-kb full-length cDNA based on a reported human genomic clone containing a partial open reading frame (ORF) coding for a novel collagen-like protein. Sequence analysis indicated that the ORF codes for the alpha(1)-chain of type XXI collagen. Assembly of the genomic data reveals a complete sequence of the human gene COL21A1. COL21A1 is localized to chromosome 6p11.2-12.3, spanning 337 kb in size. The gene contains 31 exons, in which the 5'-untranslated exons 1 and 1a are alternatively spliced. The exon/domain organization of COL21A1 resembles that of the reported FACIT collagen genes, including COL9A1, COL9A2, COL9A3, and COL19A1, suggesting that these genes may have derived from the same ancestor FACIT gene by duplication. The expression of COL21A1 in human tissues is developmentally regulated, with a higher level at fetal stages. Type XXI collagen is an extracellular matrix component of the blood vessel walls, secreted by smooth-muscle cells. Platelet-derived growth factor (PDGF) has a pronounced effect on the stimulation of COL21A1 expression in cultured aortic smooth-muscle cells, suggesting that alpha1(XXI) collagen may contribute to the extracellular matrix assembly of the vascular network during blood vessel formation.


Assuntos
Cromossomos Humanos Par 6/genética , Colágenos Associados a Fibrilas/genética , Colágenos Fibrilares/genética , Genoma Humano , Artérias/metabolismo , Colágenos Associados a Fibrilas/biossíntese , Humanos , Dados de Sequência Molecular , Músculo Liso Vascular/metabolismo , Fases de Leitura Aberta/genética , Fator de Crescimento Derivado de Plaquetas/metabolismo , Análise de Sequência de DNA
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