RESUMO
Sialic acids are important sugars at the reducing end of glycoproteins and glycolipids. They are among many other functions involved in cell-cell interactions, host-pathogen recognition and the regulation of serum half-life of glycoproteins. An important modification of sialic acids is O-acetylation, which can alter or mask the biological properties of the parent sialic acid molecule. The nature of mammalian sialate-O-acetyltransferases (EC 2.3.1.45) involved in their biosynthesis is still unknown. We have identified the human CasD1 (capsule structure1 domain containing 1) gene as a candidate to encode the elusive enzyme. The human CasD1 gene encodes a protein with a serine-glycine-asparagine-histidine hydrolase domain and a hydrophobic transmembrane domain. Expression of the Cas1 protein tagged with enhanced green fluorescent protein in mammalian and insect cells directed the protein to the medial and trans-cisternae of the Golgi. Overexpression of the Cas1 protein in combination with α-N-acetyl-neuraminide α-2,8-sialyltransferase 1 (GD3 synthase) resulted in an up to 40% increased biosynthesis of 7-O-acetylated ganglioside GD3. By quantitative real-time polymerase chain reaction, we found up to 5-fold increase in CasD1 mRNA in tumor cells overexpressing O-Ac-GD3. CasD1-specific small interfering RNA reduced O-acetylation in tumor cells. These results suggest that the human Cas1 protein is directly involved in O-acetylation of α2-8-linked sialic acids.
Assuntos
Acetiltransferases/genética , Carboidratos Epimerases/metabolismo , Ácido N-Acetilneuramínico/metabolismo , Acetilação , Acetiltransferases/metabolismo , Sequência de Aminoácidos , Domínio Catalítico , Linhagem Celular , Clonagem Molecular , Mineração de Dados , Técnicas de Silenciamento de Genes , Humanos , Linfócitos/metabolismo , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Interferência de RNA , Alinhamento de Sequência , Especificidade por Substrato , Regulação para CimaRESUMO
Viral O-acetylesterases were first identified in several viruses, including influenza C viruses and coronaviruses. These enzymes are capable of removing cellular receptors from the surface of target cells. Hence they are also known as "receptor destroying" enzymes. We have cloned and expressed several recombinant viral O-acetylesterases. These enzymes were secreted from Sf9 insect cells as chimeric proteins fused to eGFP. A purification scheme to isolate the recombinant O-acetylesterase of influenza C virus was developed. The recombinant enzymes derived from influenza C viruses specifically hydrolyze 9-O-acetylated sialic acids, while that of sialodacryoadenitis virus, a rat coronavirus related to mouse hepatitis virus, is specific for 4-O-acetylated sialic acid. The recombinant esterases were shown to specifically de-O-acetylate sialic acids on glycoconjugates. We have also expressed esterase knockout proteins of the influenza C virus hemagglutinin-esterase. The recombinant viral proteins can be used to unambiguously identify O-acetylated acids in a variety of assays.
Assuntos
Acetilesterase/química , Ácido N-Acetilneuramínico/química , Proteínas Recombinantes/química , Proteínas Virais/química , Acetilesterase/metabolismo , Animais , Baculoviridae/metabolismo , Bovinos , Membrana Celular/metabolismo , Clonagem Molecular , Coronavirus/metabolismo , Eletroforese em Gel de Poliacrilamida , Glicoconjugados/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Células HeLa , Humanos , Imunoprecipitação , Gammainfluenzavirus/metabolismo , Mucinas/metabolismo , Ácido N-Acetilneuramínico/metabolismo , Plasmídeos/metabolismo , Estrutura Terciária de Proteína , Fatores de TempoRESUMO
Infectious salmon anemia virus (ISAV) is the causative agent of infections in farmed Atlantic salmon. ISAV presumably represents a new genus within the Orthomyxoviridae. ISAV has been shown earlier to exhibit a receptor-destroying activity, which was defined as an acetylesterase with unknown specificity. We have analyzed the substrate specificity of the ISAV esterase in detail. Purified ISAV hydrolyzed free 5-N-acetyl-4-O-acetyl neuraminic acid. In addition, the purified 9-O-acetylated sialic acid derivative was also hydrolyzed, but at lower rates. When we used a glycosidically bound substrate, ISAV was unable to hydrolyze 9-O-acetylated sialic acid, which represents the major substrate for the influenza C virus esterase. ISAV completely de-O-acetylated glycoprotein-bound 5-N-acetyl-4-O-acetyl neuraminic acid. Thus, the enzymatic activity of the hemagglutinin-esterase of ISAV is comparable to that of the sialate-4-O-esterases of murine coronaviruses and related group 2 coronaviruses. In addition, we found that ISAV specifically binds to glycoproteins containing 4-O-acetylated sialic acids. Both the ISAV esterase and recombinant rat coronavirus esterase specific for 4-O-acetylated sialic acids hydrolyzed ISAV receptors on horse and rabbit erythrocytes, indicating that this sialic acid represents a receptor determinant for ISAV.