He Qinghua, Hong Jian, Zheng Yucai, Jin Suyu
College of Life Science and Technology, Southwest University for Nationalities, Chengdu, China.
J Reprod Dev. 2008 Jun;54(3):229-32. doi: 10.1262/jrd.19103. Epub 2008 Apr 14.
Lactate dehydrogenase C (LDH-C) has been reported to play a role in the energy metabolism of mammal spermatozoa. However, the functions and expression patterns of LDH-C still remain unclear. In order to elucidate the functions and expression patterns of LDH-C, we cloned the cDNA of yak LDH-C. Total RNA was extracted from yak testes and reverse transcribed and amplified by PCR. The full length open reading frame (ORF) of LDH-C and its five splice variants were obtained. The full length ORF contained 999 bp encoding a 332-amino-acid protein that showed 100% identity with bovine LDH-C. Compared with the full length ORF of LDH-C, the five variants used the same start codon as the full length ORF and encoded 5 putative proteins. In detail, variants 1 (missing the coding sequence of exon 6 and 7) and 2 (missing the coding sequence of exon 7) bear the entire nicotinamide-adenine dinucleotide (NAD) binding domain and an active site. Variants 3 (missing the first 42 nuleotides of exon 4) and 4 (missing the coding sequences of exons 5, 6 and 7) lack part of the NAD binding domain but contained the entire active site. Variant 5 (missing the coding sequence of exons 4 and 7) lacks a large part of the NAD binding domain and the entire active site. Native polyacrylamide gel electrophoresis was performed to determine if the splice variants can be translated into proteins. However, native PAGE detected no specific bands from yak testes and bovine spermatozoa. This study suggests the alternative splicing of LDH-C is ubiquitous in bovine testes and might be involved in regulation of LDH-C expression. The findings also help to elucidate the functions of LDH-C.
据报道，乳酸脱氢酶C（LDH-C）在哺乳动物精子的能量代谢中发挥作用。然而，LDH-C的功能和表达模式仍不清楚。为了阐明LDH-C的功能和表达模式，我们克隆了牦牛LDH-C的cDNA。从牦牛睾丸中提取总RNA，通过PCR进行逆转录和扩增。获得了LDH-C的全长开放阅读框（ORF）及其五个剪接变体。全长ORF包含999 bp，编码一个332个氨基酸的蛋白质，与牛LDH-C具有100%的同一性。与LDH-C的全长ORF相比，这五个变体使用与全长ORF相同的起始密码子，并编码5种假定蛋白质。具体而言，变体1（缺失外显子6和7的编码序列）和变体2（缺失外显子7的编码序列）具有完整的烟酰胺腺嘌呤二核苷酸（NAD）结合结构域和一个活性位点。变体3（缺失外显子4的前42个核苷酸）和变体4（缺失外显子5、6和7的编码序列）缺少部分NAD结合结构域，但包含完整的活性位点。变体5（缺失外显子4和7的编码序列）缺少大部分NAD结合结构域和整个活性位点。进行了非变性聚丙烯酰胺凝胶电泳以确定剪接变体是否能被翻译成蛋白质。然而，非变性PAGE在牦牛睾丸和牛精子中未检测到特异性条带。本研究表明，LDH-C的可变剪接在牛睾丸中普遍存在，可能参与LDH-C表达的调控。这些发现也有助于阐明LDH-C的功能。
