RESUMO
Nucleotides are involved in regulating a number of important processes ranging from inflammation to platelet aggregation. Enzymes that can modulate levels of nucleotides in the blood therefore represent important regulatory components in these physiological systems. CD39L4 is a soluble E-nucleoside triphosphate dephosphohydrolase (E-NTPDase) with specificity for nucleotide diphosphates (NDPs). In this study, stable mammalian and insect cell lines were generated expressing CD39L4 protein to purify and characterize the recombinant protein. We demonstrate that recombinant CD39L4 protein expressed in human embryonic carcinoma 293 cells is glycosylated by comparing the molecular masses before and after glycosidase treatment. Activity measurements of CD39L4 isolated from tunicamycin-treated, transiently transfected COS-7 cells indicate that glycosylation is not required for full ADPase activity. Recombinant human CD39L4 protein isolated from stable insect cells was glycosylated differently, but also demonstrated relative activity comparable to that of the mammalian protein. When denatured by SDS under nonreducing conditions, a fraction of the CD39L4 protein migrates as a 110 kDa disulfide-linked dimer. We determined that the monomer is the most active form of CD39L4 by measuring the activity of sucrose density gradient fractions of monomers and partially purified dimers. The physiological significance of the biochemical and enzymatic characterization is discussed.
Assuntos
Adenosina Trifosfatases/química , Adenosina Trifosfatases/metabolismo , Adenosina Trifosfatases/biossíntese , Adenosina Trifosfatases/genética , Animais , Apirase/química , Apirase/metabolismo , Células COS , Linhagem Celular , Dimerização , Dissulfetos/química , Ativação Enzimática/genética , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Humanos , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Spodoptera/genética , TransfecçãoRESUMO
The interleukin (IL)-1 family of proteins plays an important role in inflammatory and defense mechanisms. The recently characterized IL1HY1 cDNA encodes a new member of the IL-1 receptor antagonist family (IL-1ra). In this report, we describe the complete nucleotide sequence of the human IL1HY1 gene. We sequenced approximately 7,600 nucleotides and found four coding exons ranging in size from 55 to 2,288 nucleotides. The 5' untranslated region is formed by one of two alternatively used exons and one invariably present exon which also contains the region encoding the first nine amino acids of the protein. IL1HY1 and IL-1ra intron positions are well conserved within the protein-coding region, providing evidence that these genes arose from a duplication of a primordial IL-1 receptor antagonist gene.
Assuntos
Interleucinas , Proteínas/química , Proteínas/genética , Receptores de Interleucina-1/antagonistas & inibidores , Regiões 5' não Traduzidas/genética , Sequência de Aminoácidos , Sequência de Bases , Cromossomos Humanos Par 2/genética , Éxons , Duplicação Gênica , Humanos , Íntrons , Dados de Sequência Molecular , Células Tumorais CultivadasRESUMO
Interleukin-1 is a potent mediator of inflammation, involved in regulating a wide variety of physiological and cellular events. We have identified and characterized a novel member of the human interleukin-1 gene family (IL1HY1). The encoded protein demonstrates significant amino acid homology to the receptor antagonist (IL-1ra) at 52%. The gene was mapped to the long arm of chromosome 2, in close proximity to the IL-1 locus. IL1HY1 message is tightly regulated being most predominantly expressed in the skin, but also detected in the spleen, brain leukocyte, and macrophage cell types. Furthermore, the message can be induced in THP-1 cells by phorbol ester (PMA) and lipopolysaccharide (LPS) treatment.
Assuntos
Cromossomos Humanos Par 2 , Interleucinas , Proteínas/genética , Receptores de Interleucina-1/antagonistas & inibidores , Pele/imunologia , Sequência de Aminoácidos , Sequência de Bases , Encéfalo/imunologia , Linhagem Celular , Mapeamento Cromossômico , Feto , Amplificação de Genes , Biblioteca Gênica , Humanos , Proteína Antagonista do Receptor de Interleucina 1 , Leucócitos/imunologia , Macrófagos/imunologia , Dados de Sequência Molecular , Família Multigênica , Especificidade de Órgãos , Proteínas/química , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Sialoglicoproteínas/química , Sialoglicoproteínas/genética , Pele/embriologia , Baço/imunologia , Transcrição GênicaRESUMO
The human ecto-apyrase gene family consists of five reported members (CD39, CD39-L1, CD39-L2, CD39-L3, and CD39-L4). The family can be subdivided into two groups by conservation of proposed structural domains. The CD39, CD39-L1, and CD39-L3 genes all encode hydrophobic portions in their carboxy and amino termini, serving as transmembrane domains for CD39 and potentially for the other two members. CD39-L2 and CD39-L4 genes encode hydrophobic portions in their amino termini, suggesting that they might encode secreted apyrases. We demonstrate that the CD39-L4 gene encodes the first reported human secreted ecto-apyrase. COS-7 cells transfected with a CD39-L4 expression construct utilizing the naturally occurring leader peptide express recombinant protein outside of the cells. This expression can be blocked by brefeldin A, a chemical that inhibits a step in mammalian secretory pathways. We also demonstrate expression of CD39-L4 message in macrophages, suggesting that the protein is present in the circulation. Furthermore, we show that CD39-L4 is an E-type apyrase, is dependent on calcium and magnesium cations, and has high degree of specificity for NDPs over NTPs as enzymatic substrates. A potential physiological role in hemostasis and platelet aggregation is presented.
