Unidade de Recursos Genéticos, Reprodução e Melhoramento Animal, Instituto Nacional dos Recursos Biológicos L-INIA Santarém, Quinta da Fonte Boa, Vale de Santarém, Portugal.
PLoS One. 2012;7(8):e42957. doi: 10.1371/journal.pone.0042957. Epub 2012 Aug 24.
A hallmark of prion diseases or transmissible spongiform encephalopaties is the conversion of the cellular prion protein (PrP(C)), expressed by the prion gene (prnp), into an abnormally folded isoform (PrP(Sc)) with amyloid-like features that causes scrapie in sheep among other diseases. prnp together with prnd (which encodes a prion-like protein designated as Doppel), and prnt (that encodes the prion protein testis specific--Prt) with sprn (shadow of prion protein gene, that encodes Shadoo or Sho) genes, constitute the "prion gene complex". Whereas a role for prnd in the proper functioning of male reproductive system has been confirmed, the function of prnt, a recently discovered prion family gene, comprises a conundrum leading to the assumption that ruminant prnt is a pseudogene with no protein expression. The main objective of the present study was to identify Prt localization in the ram reproductive system and simultaneously to elucidate if ovine prnt gene is transcribed into protein-coding RNA. Moreover, as Prt is a prnp-related protein, the amyloid propensity was also tested for ovine and caprine Prt. Recombinant Prt was used to immunize BALB/c mice, and the anti-Prt polyclonal antibody (APPA) immune response was evaluated by ELISA and Western Blot. When tested by indirect immunofluorescence, APPA showed high avidity to the ram sperm head apical ridge subdomain, before and after induced capacitation, but did not show the same behavior against goat spermatozoa, suggesting high antibody specificity against ovine-Prt. Prt was also found in the testis when assayed by immunohistochemistry during ram spermatogenesis, where spermatogonia, spermatocytes, spermatids and spermatozoa, stained positive. These observations strongly suggest ovine prnt to be a translated protein-coding gene, pointing to a role for Prt protein in the ram reproductive physiology. Besides, caprine Prt appears to exhibit a higher amyloid propensity than ovine Prt, mostly associated with its phenylalanine residue.
朊病毒病或传染性海绵状脑病的一个标志是细胞朊蛋白 (PrP(C)) 的转化，该蛋白由朊病毒基因 (prnp) 表达，转化为具有淀粉样特征的异常折叠异构体 (PrP(Sc))，导致绵羊中的瘙痒病和其他疾病。prnp 与 prnd（编码一种称为 Doppel 的朊病毒样蛋白）以及 prnt（编码睾丸特异性朊病毒蛋白——Prt）和 sprn（朊病毒蛋白基因的阴影，编码 Shadoo 或 Sho）基因一起构成“朊病毒基因复合物”。虽然已经证实 prnd 在雄性生殖系统的正常功能中起作用，但 prnt 的功能，一个最近发现的朊病毒家族基因，构成了一个难题，导致假设反刍动物 prnt 是一个没有蛋白质表达的假基因。本研究的主要目的是确定 ram 生殖系统中 Prt 的定位，并同时阐明绵羊 prnt 基因是否转录成蛋白质编码 RNA。此外，由于 Prt 是 Prnp 的相关蛋白，因此还测试了绵羊和山羊 Prt 的淀粉样倾向。使用重组 Prt 免疫 BALB/c 小鼠，并通过 ELISA 和 Western Blot 评估抗 Prt 多克隆抗体 (APPA) 的免疫反应。当通过间接免疫荧光测试时，APPA 对 ram 精子头部顶端脊亚域表现出高亲和力，在诱导顶体反应前后均如此，但对山羊精子没有相同的行为，表明对绵羊-Prt 具有高抗体特异性。在 ram 精子发生期间通过免疫组织化学检测到 Prt 也存在于睾丸中，其中精原细胞、精母细胞、精子细胞和精子均呈阳性染色。这些观察结果强烈表明绵羊 prnt 是一种翻译的蛋白质编码基因，表明 Prt 蛋白在 ram 生殖生理学中起作用。此外，山羊 Prt 似乎比绵羊 Prt 表现出更高的淀粉样倾向，主要与它的苯丙氨酸残基有关。
