RESUMO
Full-length clones encoding a novel member of the metalloproteinase-like, disintegrin-like, cysteine-rich (MDC) family of proteins have been isolated from a monkey (Macaca fascicularis) testicular cDNA library. The encoded putative 82 kDa transmembrane protein (tMDC I) shows striking sequence similarity to other members of the MDC family including rat and monkey EAP I, guinea-pig PH-30 and human MDC protein, as well as a number of snake venom components.
Assuntos
Expressão Gênica , Macaca fascicularis/genética , Peptídeos/genética , Proteínas/genética , Testículo/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Desintegrinas , Biblioteca Gênica , Cobaias , Humanos , Masculino , Proteínas de Membrana , Metaloendopeptidases/genética , Dados de Sequência Molecular , Biossíntese de Proteínas , Sinais Direcionadores de Proteínas/biossíntese , Sinais Direcionadores de Proteínas/genética , Ratos , Homologia de Sequência de Aminoácidos , Transcrição GênicaRESUMO
Full-length clones have been isolated from a Macaca fascicularis cDNA library corresponding to a 2.65 kb mRNA present at high steady state levels in the testis. Sequence analysis suggests that the predicted translation product, tMDC II protein, represents a novel member of the metalloproteinase-like, disintegrin-like, cysteine-rich (MDC) family of mammalian proteins.
Assuntos
Cisteína/análise , Metaloendopeptidases/genética , Proteínas/genética , Testículo/enzimologia , Proteínas ADAM , Sequência de Aminoácidos , Animais , Sequência de Bases , DNA Complementar/química , DNA Complementar/isolamento & purificação , Desintegrinas , Biblioteca Gênica , Macaca fascicularis , Masculino , Metaloendopeptidases/química , Dados de Sequência Molecular , Peptídeos/genética , Proteínas/química , RNA Mensageiro/química , Alinhamento de SequênciaRESUMO
Increasing number of sequence-related cysteine-rich membrane proteins containing metalloproteinase-like and disintegrin-like domains (the MDC protein family) have been identified in mammalian tissues. Here, we report the cloning and sequence analysis of cDNAs encoding several rat orthologues of this protein family, some of which are found to be expressed exclusively in the male reproductive tract, others exhibiting a broader tissue distribution. We also examine their expression in prepubertal an adult rat testis, which, in conjunction with the data on tissue distribution, form a necessary prelude to further studies aimed at establishing their individual functions.
Assuntos
Proteínas de Membrana/metabolismo , Maturidade Sexual/fisiologia , Testículo/metabolismo , Proteínas ADAM , Sequência de Aminoácidos , Animais , Clonagem Molecular , Fertilinas , Humanos , Masculino , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Proteínas de Membrana/genética , Metaloendopeptidases/genética , Metaloendopeptidases/metabolismo , Dados de Sequência Molecular , Proteínas/genética , Proteínas/metabolismo , Ratos , Análise de Sequência de DNA , Testículo/crescimento & desenvolvimentoRESUMO
All members of the MDC family of integral membrane proteins contain a metalloproteinase-like domain, a disintegrin-like domain and a cysteine-rich domain. They have been identified in a wide range of mammalian tissues and many are abundantly expressed in the male reproductive tract. We have previously cloned and sequenced several MDC transcripts expressed in the testes of the macaque (Macaca fascicularis). Using polyclonal antisera we have localized a number of these MDC proteins (fertilin alpha, fertilin beta, tMDC I, tMDC II and tMDC III) to spermatogenic cells and demonstrated that they are processed as spermatozoa pass through the epididymis, yielding proteins that retain their disintegrin domain on mature, fertilization-competent cauda spermatozoa. In rodents, two of these MDC proteins (fertilin beta and tMDC I) have been implicated in egg recognition, mediated by a disintegrin-integrin interaction. It is possible that the additional MDC proteins expressed on macaque spermatozoa may also be involved in this process.
Assuntos
Epididimo/metabolismo , Proteínas de Membrana/metabolismo , Transporte Espermático/fisiologia , Espermatozoides/metabolismo , Animais , Western Blotting , Macaca fascicularis , Masculino , Proteínas de Membrana/análise , Distribuição TecidualRESUMO
Fertilin alpha and beta are members of the MDC (metalloproteinase-like, disintegrin-like, cysteine-rich) protein family and are expressed on the sperm surface where they have been proposed to play a role in mammalian fertilization. Inhibition of sperm-oocyte binding and sperm-oocyte fusion make fertilin an attractive target for the development of an immunocontraceptive vaccine. Full-length cDNAs encoding alpha and beta fertilin subunits were isolated from a rat testis cDNA library and sequenced. Using reverse transcription-polymerase chain reaction (RT-PCR), the developmental expression of fertilin alpha and beta was determined in pre-pubertal and mature rat testes. Fertilin alpha mRNA was present at all stages of development, suggesting that it is not exclusively expressed in post-meiotic germ cells. In contrast, fertilin beta mRNA was first identified in day 19 testes, coincident with the presence of pachytene spermatocytes. Polyclonal antisera raised against a 28-residue peptide (corresponding to part of the disintegrin domain) and two recombinant fusion proteins identified a 90 kDa protein in testicular sperm extracts and a 60 kDa protein in caput and cauda epididymidal sperm extracts, the predicted sizes for rat fertilin beta precursor and mature protein respectively. Indirect immunofluorescence using the anti-peptide antisera stained the acrosomal cap of permeabilized testicular, caput and caudal spermatozoa and elongating spermatids in testicular sections.
Assuntos
Glicoproteínas de Membrana/genética , Metaloendopeptidases/genética , Proteínas ADAM , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , DNA Complementar/análise , DNA Complementar/química , DNA Complementar/genética , Fertilinas , Regulação da Expressão Gênica no Desenvolvimento , Imuno-Histoquímica , Masculino , Dados de Sequência Molecular , Processamento de Proteína Pós-Traducional , Ratos , Ratos Wistar , Reprodução/genética , Reprodução/fisiologia , Análise de Sequência , Homologia de Sequência de Aminoácidos , Testículo/química , Transcrição GênicaRESUMO
A large number of sequence-related, cysteine-rich membrane proteins containing metalloproteinase-like and disintegrin-like domains (the MDC protein family) have been identified in mammalian tissues from a variety of species. Previous studies in the macaque (Macaca fascicularis) have led to the cDNA cloning and sequence analysis of a number of MDC proteins which are abundantly expressed in the male reproductive tract. We now describe the distribution of seven of these macaque MDC transcripts in a range of different tissues. This description includes a novel macaque testis-derived MDC, tMDC III, whose full-length sequence is reported for the first time. In addition, polyclonal antisera have been used to localize a number of these MDC proteins to spermatogenic cells in testis sections, and to demonstrate their processing on the sperm surface during epididymal transit.