Bioinformatic Study On The Structure Of Human Lactate Dehydrogenase C4 And Construction Of Recombinant Vectors For The Expression Of The L178X Mutant

Sperm-specific lactate dehydrogenase, generally consisting of four copies of subunit C, is also called lactate dehydrogenase C4, or LDH-C4. Being a key enzyme for sperm energy metabolism, it is closely related to spermatogenesis, spermatic metabolism and sperm capacitation. We found a nonsense mutation L178X in the process of the study of the relationship between LDHC gene mutation and male infertility.To study the influences of preciously-found L178X mutation on the structure and function of LDH-C4, I-TASSER was used to predict the structure and function of LDH-C4. We found that both LDH-C4 and somatic LDH belong to LHD-1 enzyme family, sharing most of the conserverd amino acids. Meanwhile, three basic characteristics were found with LDH-1 enzyme family: NAD binding site, substrate binding site, as well as interface of LDHC subunit.Based on the analysis above, we prepared a prokaryotic expression vector pET-28a(+)-hLDHC/L178X, and found that the L178X mutant had no LDH activity.Furthermore, we constructed a eukaryotic expression vector pVAX1-hLDHC/L178X to perform transcription/translation assay in vitro to access the activity of the wild type, heterozygous L178X mutant and homozygous L178X mutant of LDH-C4. We found that the activity of the wild-type LDH-C4 was twice as the heterozygote.In a word, our studies lay a foundation for illuminating the influence of the LDHC gene mutation on the function of LDH-C4.