Comparison of Prostate‐Specific Transglutaminase Function in the Great Apes
Authors: Ports Brianna L. , Jensen‐Seaman Michael 
Published: May 2022

The copulatory plug is a hardened structure formed through semen coagulation post‐ejaculation which is theorized to protect against remating, stimulate the female reproductive tract, and aid in sperm release and transport. This structure is prevalent in species that experience high levels of sperm competition including chimpanzees and several other primates. Chimpanzees experience high levels of sperm competition due to their polygynandrous mating system. In polygynandry females will mate with multiple partners during an estrus cycle leading to direct competition between the sperm of each male to fertilize the same egg. This varies distinctly from polygynous and monogamous species, such as humans, where females mate with only one male and sperm competition is diminished. Based on previous work in rats, prostate‐specific transglutaminase 4 (TGM4) is implicated as the driving force of the formation of the copulatory plug. TGM4 is a member of the transglutaminase family of enzymes which catalyze the calcium‐dependent formation of an isopeptide bond between glutamine and lysine residues. While humans do not experience a great deal of sperm competition or form a copulatory plug, human semen goes through a similar process of post‐ejaculatory coagulation which is predicted to be carried out by TGM4 under the same mechanism. Interestingly, there are only seven amino acid differences between the human and chimpanzee TGM4s and yet the degree of coagulation between the two species is vastly different. The aim of this study to characterize and compare the function of these proteins to determine the major contributing factor in semen coagulation. The primary hypothesis was that chimpanzee TGM4 would have higher enzymatic activity and more readily cross‐link artificial substrates than human TGM4. To test this hypothesis, the coding sequences were acquired for human and chimpanzee TGM4 as well as eight other primate species. Using a maximum likelihood‐based method, we generated a gene tree of primate TGM4 and calculated dN/dS ratios for each branch. Human TGM4 had a dN/dS ratio of 1.04 suggesting neutral evolution while chimpanzee TGM4 had a ratio of >1 suggesting strong positive selection. We further predicted the sequence of TGM4 of the last human‐chimp ancestor using ancestral sequence reconstruction which utilizes the known phylogenetic tree and a maximum likelihood approach with the software package Phylogenetic Analysis by Maximum Likelihood (PAML). Coding sequences representing TGM4 from humans, chimpanzees, and the last human‐chimp ancestor were cloned into a mammalian expression vector containing a C‐terminal His6tag and were transfected into HEK‐293T cells. Purified TGM4 was used in an in‐vitro colorimetric enzymatic assay to quantify enzymatic activity. Our results suggest that mating system variation has led to differences in both sequence and function of seminal proteins. These functional differences speak to the impact of even a small number of mutations on protein function as well as the influence of environmental pressures on protein evolution.