Assuntos
Exostose Múltipla Hereditária/genética , Genes Supressores de Tumor , Heparitina Sulfato/fisiologia , N-Acetilglucosaminiltransferases/fisiologia , Proteínas/fisiologia , Acetilglucosamina/metabolismo , Adulto , Animais , Biopolímeros , Desenvolvimento Ósseo/genética , Neoplasias Ósseas/epidemiologia , Neoplasias Ósseas/genética , Células CHO , Cartilagem/patologia , Criança , Cricetinae , Análise Mutacional de DNA , Evolução Molecular , Genes Dominantes , Genes Letais , Teste de Complementação Genética , Predisposição Genética para Doença , Ácido Glucurônico/metabolismo , Glicosilação , Heparitina Sulfato/biossíntese , Humanos , Invertebrados/crescimento & desenvolvimento , Invertebrados/metabolismo , Substâncias Macromoleculares , Camundongos , Camundongos Knockout , Modelos Biológicos , N-Acetilglucosaminiltransferases/deficiência , N-Acetilglucosaminiltransferases/genética , Proteína Relacionada ao Hormônio Paratireóideo , Proteínas/genética , Proteínas/metabolismo , Receptor Tipo 1 de Hormônio Paratireóideo , Receptores de Hormônios Paratireóideos/metabolismo , Risco , Relação Estrutura-Atividade , Vertebrados/crescimento & desenvolvimento , Vertebrados/metabolismoRESUMO
Hereditary multiple exostoses (HME), a dominantly inherited genetic disorder characterized by multiple cartilaginous tumors, is caused by mutations in members of the EXT gene family, EXT1 or EXT2. The corresponding gene products, exostosin-1 (EXT1) and exostosin-2 (EXT2), are type II transmembrane glycoproteins which form a Golgi-localized heterooligomeric complex that catalyzes the polymerization of heparan sulfate (HS). Although the majority of the etiological mutations in EXT are splice-site, frameshift, or nonsense mutations that result in premature termination, 12 missense mutations have also been identified. Furthermore, two of the reported etiological missense mutations (G339D and R340C) have been previously shown to abrogate HS biosynthesis (McCormick et al. 1998). Here, a functional assay that detects HS expression on the cell surface of an EXT1-deficient cell line was used to test the remaining missense mutant exostosin proteins for their ability to rescue HS biosynthesis in vivo. Our results show that EXT1 mutants bearing six of these missense mutations (D164H, R280G/S, and R340S/H/L) are also defective in HS expression, but surprisingly, four (Q27K, N316S, A486V, and P496L) are phenotypically indistinguishable from wild-type EXT1. Three of these four "active" mutations affect amino acids that are not conserved among vertebrates and invertebrates, whereas all of the HS-biosynthesis null mutations affect only conserved amino acids. Further, substitution or deletion of each of these four residues does not abrogate HS biosynthesis. Taken together, these results indicate that several of the reported etiological mutant EXT forms retain the ability to synthesize and express HS on the cell surface. The corresponding missense mutations may therefore represent rare genetic polymorphisms in the EXT1 gene or may interfere with as yet undefined functions of EXT1 that are involved in HME pathogenesis.
Assuntos
Exostose Múltipla Hereditária/enzimologia , Exostose Múltipla Hereditária/genética , N-Acetilglucosaminiltransferases/genética , N-Acetilglucosaminiltransferases/metabolismo , Mutação Puntual/genética , Sequência de Aminoácidos , Animais , Células CHO , Cromatografia por Troca Iônica , Cricetinae , Fator 2 de Crescimento de Fibroblastos/metabolismo , Teste de Complementação Genética , Heparitina Sulfato/metabolismo , Humanos , Dados de Sequência Molecular , Mutação de Sentido Incorreto/genética , N-Acetilglucosaminiltransferases/biossíntese , N-Acetilglucosaminiltransferases/química , Polimorfismo Genético/genética , Alinhamento de SequênciaRESUMO
To gain entry into the host, viruses use host cell surface molecules that normally serve as receptors for other ligands. Herpes simplex virus type 1 (HSV-1) uses heparan sulphate (HS) glycosaminoglycans (GAGs) as receptors for initial attachment to the host cell surface. HS GAGs are both ubiquitous and structurally diverse, and normally serve as critical mediators of interactions between the cell and the extracellular environment. We have used the HS binding ability of HSV-1 to identify the function of a cellular gene, EXT1, which is involved in HS polymerisation. Cellular factors that affect virus growth and replication are often key regulators of the cell cycle and EXT1 is no different-humans with inherited mutations in EXT1 have developmental defects that lead to bone tumours (hereditary multiple exostoses, HME) and sometimes chondrosarcomas. Thus, as a result of using HSV-1 as a molecular probe, a functionally orphaned disease gene now has a defined function. These findings highlight the utility of viruses for investigating important cellular processes.
Assuntos
Exostose Múltipla Hereditária/genética , Heparitina Sulfato/metabolismo , Herpesvirus Humano 1/fisiologia , Linhagem Celular , Exostose Múltipla Hereditária/etiologia , Genes Supressores de Tumor , Herpesvirus Humano 1/metabolismo , Humanos , Sondas Moleculares , Mutação , N-Acetilglucosaminiltransferases/genética , Replicação ViralRESUMO
We found that chronic lymphocytic leukemic (CLL) B cells are highly sensitive to infection with vectors derived from replication-defective herpes simplex virus-1 (rdHSV-1). CLL B cells were found to express high levels of herpes virus entry mediator (Hve) A, but not HveC, the other known receptor for HSV-1. An HveA cDNA from CLL cells was found to encode Arg-->Lys and Val-->Iso substitutions at amino acids 17 and 241, respectively. Nevertheless, this cDNA encoded a functional receptor for HSV-1 when transfected into Chinese hamster ovarian (CHO) cells. Antibodies to HveA could block rdHSV-1 infection of CLL cells and HveA-transfected CHO cells with similar efficiencies in vitro. In contrast to B cells of normal donors, CLL B cells were resistant to the cytopathic effects of infection by rdHSV-1 and maintained high-level expression of the transgene for several days in vitro. We propose that this is due to the expression by CLL cells of the anti-apoptotic protein, bcl-2. Consistent with this, we found that transduction of HeLa cells with a retrovirus expression vector encoding bcl-2 rendered HeLa cells resistant to the cytopathic effects of rdHSV-1. HSV-1-derived vectors should be excellent vehicles for gene transfer into CLL B cells, allowing for its potential use in gene therapy for this disease. Gene Therapy (2000) 7, 1210-1216.
Assuntos
Herpes Simples/virologia , Leucemia Linfocítica Crônica de Células B/virologia , Animais , Transformação Celular Viral , Células Clonais , Cricetinae , Citometria de Fluxo , Genes bcl-2 , Herpes Simples/enzimologia , Herpes Simples/genética , Humanos , Leucemia Linfocítica Crônica de Células B/enzimologia , Leucemia Linfocítica Crônica de Células B/genética , Células Tumorais Cultivadas , beta-Galactosidase/metabolismoRESUMO
G207 is a conditionally replicating derivative of herpes simplex virus (HSV) type-1 strain F engineered with deletions of both gamma(1)34.5 loci and a lacZ insertion disabling the UL39 gene. We have demonstrated the efficacy of G207 in treating malignant glial tumors in athymic mice, as well as the safety of intracerebral G207 inoculation in mice and in Aotus nancymai. We sought to determine the safety of G207 inoculation into cerebral malignant glial tumors in humans. Criteria for inclusion into this dose-escalation study were the diagnosis of histologically proven malignant glioma, Karnofsky score > or = 70, recurrence despite surgery and radiation therapy, and an enhancing lesion greater than 1 cm in diameter. Serial magnetic resonance images were obtained for volumetric analysis. The trial commenced at a dose of 10(6) plaque forming units (p.f.u.) inoculated at a single enhancing site and was completed when the 21st patient was inoculated with 3x10(9) p.f.u. at five sites. While adverse events were noted in some patients, no toxicity or serious adverse events could unequivocally be ascribed to G207. No patient developed HSV encephalitis. We found radiographic and neuropathologic evidence suggestive of anti-tumor activity and long-term presence of viral DNA in some cases.
Assuntos
Neoplasias Encefálicas/terapia , Terapia Genética/métodos , Glioblastoma/terapia , Herpesvirus Humano 1/crescimento & desenvolvimento , Recidiva Local de Neoplasia/terapia , Replicação Viral , Adulto , Idoso , Anticorpos Antivirais/sangue , Neoplasias Encefálicas/patologia , Neoplasias Encefálicas/virologia , Progressão da Doença , Feminino , Seguimentos , Glioblastoma/patologia , Glioblastoma/virologia , Herpesvirus Humano 1/genética , Herpesvirus Humano 1/imunologia , Humanos , Imageamento por Ressonância Magnética , Masculino , Pessoa de Meia-Idade , Recidiva Local de Neoplasia/patologia , Recidiva Local de Neoplasia/virologia , Taxa de Sobrevida , Resultado do TratamentoRESUMO
Recent advances in clarifying the molecular mechanisms involved in Ag processing and presentation have relied heavily on the use of somatic cell mutants deficient in proteasome subunits, TAP transporter, and cell surface expression of MHC class I molecules. Of particular interest currently are those mutants that lack specific protease activity involved in the generation of antigenic peptides. It is theoretically possible that deficiencies of this nature could selectively prevent the cleavage of certain peptide bonds and thus generate only a subset of antigenic peptides. Gro29/Kb cell line is derived from the wild-type murine Ltk- cell line. This cell line is one example of a mutant that lacks specific protease activities. This deficiency manifests itself in an inability to generate a subset of immunodominant peptide epitopes derived from vesicular stomatitis virus and herpes simplex virus. This in turn leads to a general inability to present these viral epitopes to cytotoxic T lymphocytes (CTL). These studies describe a unique Ag processing deficiency and provide new insight into the role of proteasome-independent proteases in MHC class I-restricted peptide generation.
Assuntos
Apresentação de Antígeno/imunologia , Epitopos de Linfócito T/metabolismo , Epitopos Imunodominantes/metabolismo , Proteínas do Nucleocapsídeo , Linfócitos T Citotóxicos/metabolismo , Linfócitos T Citotóxicos/virologia , Membro 2 da Subfamília B de Transportadores de Cassetes de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/biossíntese , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Animais , Apresentação de Antígeno/genética , Antígenos Virais/metabolismo , Cisteína Endopeptidases/biossíntese , Regulação da Expressão Gênica/imunologia , Antígenos H-2/biossíntese , Antígenos H-2/genética , Células L , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Complexos Multienzimáticos/biossíntese , Nucleocapsídeo/deficiência , Nucleocapsídeo/genética , Nucleocapsídeo/imunologia , Nucleocapsídeo/metabolismo , Peptídeos/imunologia , Peptídeos/metabolismo , Complexo de Endopeptidases do Proteassoma , Receptores Proteína Tirosina Quinases/genética , Simplexvirus/imunologia , Linfócitos T Citotóxicos/imunologia , Vírus da Estomatite Vesicular Indiana/imunologia , Proteínas do Envelope Viral/deficiência , Proteínas do Envelope Viral/genéticaRESUMO
Hereditary multiple exostoses, a dominantly inherited genetic disorder characterized by multiple cartilaginous tumors, is caused by mutations in members of the EXT gene family, EXT1 or EXT2. The proteins encoded by these genes, EXT1 and EXT2, are endoplasmic reticulum-localized type II transmembrane glycoproteins that possess or are tightly associated with glycosyltransferase activities involved in the polymerization of heparan sulfate. Here, by testing a cell line with a specific defect in EXT1 in in vivo and in vitro assays, we show that EXT2 does not harbor significant glycosyltransferase activity in the absence of EXT1. Instead, it appears that EXT1 and EXT2 form a hetero-oligomeric complex in vivo that leads to the accumulation of both proteins in the Golgi apparatus. Remarkably, the Golgi-localized EXT1/EXT2 complex possesses substantially higher glycosyltransferase activity than EXT1 or EXT2 alone, which suggests that the complex represents the biologically relevant form of the enzyme(s). These findings provide a rationale to explain how inherited mutations in either of the two EXT genes can cause loss of activity, resulting in hereditary multiple exostoses.
Assuntos
Complexo de Golgi/metabolismo , Heparitina Sulfato/biossíntese , N-Acetilglucosaminiltransferases , Proteínas/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Catálise , Bovinos , Linhagem Celular , Exostose Múltipla Hereditária/genética , Genes Supressores de Tumor/genética , Glicosiltransferases/metabolismo , Proteínas de Fluorescência Verde , Células HeLa , Humanos , Células L , Proteínas Luminescentes/genética , Substâncias Macromoleculares , Camundongos , Microscopia de Fluorescência , Dados de Sequência Molecular , Mutação , Proteínas/química , Proteínas/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismoRESUMO
Attenuated viral vectors based on herpes simplex virus (HSV) are capable of killing cancer cells directly while sparing normal tissue in animal models of disease. This selective ability is likely due to the evolutionary constraints on the virus to establish lifelong infection in its host without causing destruction of normal tissues. However, extensive experimental animal data show that cancer cells are able to sustain a productive viral infection, which ultimately leads to cell death and tumour regression. Moreover, preliminary results generated in two Phase I clinical studies of modified replicating HSV for the treatment of brain tumours (e.g., glioblastoma multiforme) have been encouraging and suggest that the safety data generated in animals are predictive of human safety. Although much progress has been made in developing oncolytic HSV vectors for clinical use, there is still a long way to go to determine which combinations of virus, surgery, radiation and chemotherapy will provide improved therapy for the control and eradication of a variety of human cancers.
Assuntos
Terapia Genética , Vetores Genéticos , Neoplasias/terapia , Simplexvirus/genética , Animais , Humanos , Simplexvirus/fisiologia , Replicação ViralRESUMO
Herpes simplex virus (HSV) is a new platform for gene therapy. We cloned the human herpesvirus HSV-1 strain F genome into a bacterial artificial chromosome (BAC) and adapted chromosomal gene replacement technology to manipulate the viral genome. This technology exploits the power of bacterial genetics and permits generation of recombinant viruses in as few as 7 days. We utilized this technology to delete the viral packaging/cleavage (pac) sites from HSV-BAC. HSV-BAC DNA is stable in bacteria and the pac-deleted HSV-BAC (p45-25) is able to package amplicon plasmid DNA as efficiently as a comparable pac-deleted HSV cosmid set when transfected into mammalian cells. Moreover, the utility of bacterial gene replacement is not limited to HSV, since most herpesviruses can be cloned as BACs. Thus, this technology will greatly facilitate genetic manipulation of all herpesviruses for their use as research tools or as vectors in gene therapy.
Assuntos
Deleção de Genes , Técnicas de Transferência de Genes , Vetores Genéticos/genética , Herpesvirus Humano 1/genética , Animais , Chlorocebus aethiops , Cromossomos Bacterianos , Genoma Viral , Mutagênese Sítio-Dirigida , Células VeroRESUMO
Bone development is a highly regulated process sensitive to a wide variety of hormones, inflammatory mediators and growth factors. One of the most common hereditary skeletal dysplasias, hereditary multiple exostoses (HME), is an autosomal dominant disorder characterized by skeletal malformations that manifest as bony, benign tumours near the end of long bones. HME is usually caused by defects in either one of two genes, EXT1 and EXT2, which encode enzymes that catalyse the biosynthesis of heparan sulphate, an important component of the extracellular matrix. Thus, HME-linked bone tumours, like many other skeletal dysplasias, probably result from disruptions in cell surface architecture. However, despite the recent success in unravelling functions for several members of the EXT gene family, significant challenges remain before this knowledge can be used to develop new approaches for the diagnosis and treatment of disease.
Assuntos
Neoplasias Ósseas/genética , Exostose Múltipla Hereditária/genética , Heparitina Sulfato/fisiologia , N-Acetilglucosaminiltransferases , Proteínas/genética , Transativadores , Idoso , Animais , Biglicano , Desenvolvimento Ósseo/genética , Cromossomos Humanos Par 8/genética , Proteínas da Matriz Extracelular , Feminino , Genes Supressores de Tumor , Predisposição Genética para Doença , Proteínas Hedgehog , Heparitina Sulfato/biossíntese , Humanos , Síndrome de Langer-Giedion/genética , Perda de Heterozigosidade , Masculino , Camundongos , Camundongos Knockout , Proteína Relacionada ao Hormônio Paratireóideo , Proteínas/fisiologia , Proteoglicanas/deficiência , Proteoglicanas/genética , Proteoglicanas/metabolismoRESUMO
The use of herpes simplex virus (HSV) vectors for gene delivery to skeletal muscle is hampered by a maturation-dependent loss of muscle fiber infectivity. Previous studies of HSV type 1 (HSV-1) infection in the rodent show that the loss of infectivity may be due, at least in part, to the development of the basal lamina throughout the course of maturation, which may block the initial events in HSV infection. To initiate infection, HSV normally attaches to cell surface heparan sulfate, which stabilizes the virus such that it can interact with secondary protein receptors required for entry into host cells. In this study, we demonstrate that heparan sulfate biosynthesis is downregulated during skeletal muscle maturation. When myofibers were treated with a variety of enzymes, including collagenase type IV or chondroitin ABC lyase, HSV infection was restored, which suggests that virus secondary receptors were present but not readily accessible to the virus in the intact myofiber. Surprisingly, we also found that HSV-1 infectivity could be restored in vitro and in vivo by exposing myofibers to low concentrations of the glycosaminoglycan analog dextran sulfate, which appears to act as a surrogate receptor to stabilize the virus at the myofiber surface such that HSV can engage additional receptors. This demonstration that the basal lamina is not an absolute block to HSV-1 infection is remarkable because it allows for the nondestructive targeting of HSV-1 to mature myofibers and greatly expands the usefulness of HSV as a gene therapy vector for the treatment of inherited and acquired diseases.
Assuntos
Sulfato de Dextrana , Terapia Genética/métodos , Vetores Genéticos/administração & dosagem , Músculo Esquelético/virologia , Receptores Virais/genética , Simplexvirus/genética , Envelhecimento , Animais , Cromatografia Líquida de Alta Pressão , Técnicas de Transferência de Genes , Camundongos , Camundongos Endogâmicos BALB C , Microscopia de Fluorescência , Integração ViralRESUMO
Gene therapy in the anterior and posterior segment tissues may have the potential to favorably influence aqueous hydrodynamics and retinal ganglion cell biology, thereby preventing, delaying, or minimizing glaucomatous damage to the optic nerve. We demonstrated the feasibility of using a herpes viral vector (ribonucleotide reductase defective HSV-1, hrR3) to deliver the lacZ reporter gene to living cat and rat eyes. Cats received injections into the anterior chamber and rats into the vitreous cavity. In cats, lacZ expression was detectable at 1 to 2 days in the anterior outer portion of the ciliary muscle and the lining of the intertrabecular spaces of the corneoscleral and uveal meshwork. Rat eyes showed lacZ expression in the retinal pigment epithelium and photoreceptor outer segments 2 days after injection.
Assuntos
Terapia Genética , Vetores Genéticos , Glaucoma/terapia , Herpesvirus Humano 1/genética , Óperon Lac/genética , Animais , Segmento Anterior do Olho/metabolismo , Segmento Anterior do Olho/patologia , Gatos , DNA Viral/genética , Feminino , Seguimentos , Expressão Gênica/efeitos dos fármacos , Técnicas de Transferência de Genes , Genes Reporter , Glaucoma/genética , Glaucoma/patologia , Células Fotorreceptoras/metabolismo , Células Fotorreceptoras/patologia , Epitélio Pigmentado Ocular/metabolismo , Epitélio Pigmentado Ocular/patologia , Ratos , Ratos Long-Evans , beta-Galactosidase/biossíntese , beta-Galactosidase/genéticaRESUMO
Hereditary multiple exostoses, characterized by multiple cartilaginous tumors, is ascribed to mutations at three distinct loci, denoted EXT1-3. Here, we report the purification of a protein from bovine serum that harbored the D-glucuronyl (GlcA) and N-acetyl-D-glucosaminyl (GlcNAc) transferase activities required for biosynthesis of the glycosaminoglycan, heparan sulfate (HS). This protein was identified as EXT2. Expression of EXT2 yielded a protein with both glycosyltransferase activities. Moreover, EXT1, previously found to rescue defective HS biosynthesis (McCormick, C., Leduc, Y., Martindale, D., Mattison, K., Esford, L. E., Dyer, A. P., and Tufaro, F. (1998) Nat. Genet. 19, 158-161), was shown to elevate the low GlcA and GlcNAc transferase levels of mutant cells. Thus at least two members of the EXT family of tumor suppressors encode glycosyltransferases involved in the chain elongation step of HS biosynthesis.
Assuntos
Glicosiltransferases/metabolismo , Heparitina Sulfato/biossíntese , N-Acetilglucosaminiltransferases , Proteínas/metabolismo , Sequência de Aminoácidos , Animais , Desenvolvimento Ósseo , Células COS , Bovinos , Clonagem Molecular , Exostose Múltipla Hereditária/metabolismo , Genes Supressores de Tumor , Humanos , Dados de Sequência Molecular , Proteínas/genéticaRESUMO
Varicella-zoster virus (VZV) interacts with cell surface heparan sulfate proteoglycans during virus attachment. In the present study, we investigated the potential involvement of two VZV glycoproteins, gB and gE, in the virus adsorption process. We showed that gB, but not gE, binds specifically to cellular heparan sulfate proteoglycans (HSPGs). Indeed, soluble recombinant gB protein (recgB) was found to bind to immobilized heparin and to MRC5 and L cells, a binding which was inhibited by heparin. Furthermore, recgB binding to two heparan sulfate-minus mutant L cell lines, gro2C and sog9 cells, was markedly reduced as compared to the parental L strain. Under the same experimental conditions, soluble recombinant VZV gE protein did not interact with heparin or with cell surfaces. We also demonstrated that the gB-HSPGs interactions were relevant to the VZV attachment to cells. Indeed, although polyclonal antibodies directed to gB did not impair the VZV binding, recgB could delay the virus adsorption. Our results thus strongly suggest that the interactions between gB and heparan sulfate proteoglycans take part in the initial VZV attachment to cell surfaces.
Assuntos
Proteoglicanas de Heparan Sulfato/metabolismo , Herpesvirus Humano 3/metabolismo , Proteínas do Envelope Viral/metabolismo , Animais , Linhagem Celular , Membrana Celular/virologia , Humanos , Camundongos , Proteínas Recombinantes/metabolismoRESUMO
Hereditary multiple exostoses (HME) is an autosomal dominant disorder characterized by the formation of cartilage-capped tumours (exostoses) that develop from the growth plate of endochondral bone. This condition can lead to skeletal abnormalities, short stature and malignant transformation of exostoses to chondrosarcomas or osteosarcomas. Linkage analyses have identified three different genes for HME, EXT1 on 8q24.1, EXT2 on 11p11-13 and EXT3 on 19p (refs 6-9). Most HME cases have been attributed to missense or frameshift mutations in these tumour-supressor genes, whose functions have remained obscure. Here, we show that EXT1 is an ER-resident type II transmembrane glycoprotein whose expression in cells results in the alteration of the synthesis and display of cell surface heparan sulfate glycosaminoglycans (GAGs). Two EXT1 variants containing aetiologic missense mutations failed to alter cell-surface glycosaminoglycans, despite retaining their ER-localization.
Assuntos
Regulação da Expressão Gênica , Genes Supressores de Tumor , Heparitina Sulfato/biossíntese , N-Acetilglucosaminiltransferases , Proteínas/fisiologia , Animais , Linhagem Celular , Cromossomos Humanos Par 11 , Cromossomos Humanos Par 8 , Exostose Múltipla Hereditária/genética , Ligação Genética , Heparitina Sulfato/genética , Humanos , Camundongos , Peso Molecular , Proteínas/genética , Propriedades de SuperfícieRESUMO
CD44 is a widely expressed cell adhesion molecule that binds the extracellular matrix component, hyaluronan, in a tightly regulated manner. Previous studies have shown that the CD44-hyaluronan interaction is affected by changes in the glycosylation state of CD44. In this study, we take advantage of several well-characterized murine L cell mutants defective in heparan sulfate synthesis (gro2C cells), heparan sulfate and chondroitin sulfate synthesis (sog9 cells), and glycosaminoglycan and oligosaccharide processing (sog8 cells) to assess the effects of these defects on the hyaluronan binding ability of CD44. In parental L cells and gro2C cells, CD44 was induced to bind hyaluronan after addition of the activating, anti-CD44 monoclonal antibody, IRAWB 14. By contrast, no inducible binding was observed in sog9 cells. Treatment of L cells with sodium chlorate, an inhibitor of sulfation, also abolished inducible hyaluronan binding. However, inducible and some constitutive hyaluronan binding was observed in sog8 cells. This indicates that sulfation and, in particular, the addition of chondroitin sulfate are required for inducible hyaluronan binding by CD44 in L cells. However, in the absence of fully processed oligosaccharides, chondroitin sulfate is not essential for hyaluronan binding, indicating that the effect of chondroitin sulfate is dependent upon the glycosylation state of the cell. Thus, in addition to glycosylation, chondroitin sulfate biosynthesis is an important post-translational modification that can affect the hyaluronan binding ability of CD44.
Assuntos
Sulfatos de Condroitina/fisiologia , Glicosaminoglicanos/biossíntese , Glicosaminoglicanos/deficiência , Receptores de Hialuronatos/metabolismo , Ácido Hialurônico/metabolismo , Células 3T3 , Animais , Anticorpos Monoclonais/metabolismo , Sítios de Ligação de Anticorpos/genética , Glicosaminoglicanos/genética , Glicosilação , Receptores de Hialuronatos/imunologia , Células L , Camundongos , Mutação , Ligação Proteica/genéticaRESUMO
It is unclear how polyglutamine expansion is associated with the pathogenesis of Huntington disease (HD). Here, we provide evidence that polyglutamine expansion leads to the formation of large intracellular aggregates in vitro and in vivo. In vitro these huntingtin-containing aggregates disrupt normal cellular architecture and increase in frequency with polyglutamine length. Huntingtin truncated at nucleotide 1955, close to the caspase-3 cleavage site, forms perinuclear aggregates more readily than full-length huntingtin and increases the susceptibility of cells to death following apoptotic stimuli. Further truncation of huntingtin to nucleotide 436 results in both intranuclear and perinuclear aggregates. For a given protein size, increasing polyglutamine length is associated with increased cellular toxicity. Asymptomatic transgenic mice expressing full-length huntingtin with 138 polyglutamines form exclusively perinuclear aggregates in neurons. These data support the hypothesis that proteolytic cleavage of mutant huntingtin leads to the development of aggregates which compromise cell viability, and that their localization is influenced by protein length.
Assuntos
Doença de Huntington/genética , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/metabolismo , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Peptídeos , Animais , Agregação Celular , Linhagem Celular , Sobrevivência Celular , Haplorrinos , Humanos , Proteína Huntingtina , Camundongos , Camundongos Transgênicos , Proteínas do Tecido Nervoso/biossíntese , Neurônios/citologia , Neurônios/metabolismo , Neurônios/patologia , Proteínas Nucleares/biossíntese , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , TransfecçãoRESUMO
Complement provides a key immunologic defense against invading pathogens; thus, a clear understanding of the interactions between cytomegalovirus (CMV) and complement may permit the development of strategies to enhance CMV neutralization. In the presence of specific anti-CMV antibodies, complement enhanced the neutralizing ability of serum by 2- to 3-fold. However, in the absence of specific anti-CMV antibodies, complement was ineffective in neutralizing CMV virions by plaque assay. Although complement alone did not mediate any neutralizing effect, CMV consumed complement activity from seronegative serum, resulting in the deposition of C3 on the virion. However, only in the presence of specific anti-CMV antibody did complement activation continue to the deposition of C9 on the virions. These results strongly suggest complement regulation by CMV virions that is modulated by anti-CMV antibody; this regulation may be attributed to three host complement regulators on the virions: CD55, CD46, and CD59.