Functional Analysis of KLK3/PSA Protease Activity in Primates

Abstract

Male reproductive proteins have been the focus of evolutionary biologists due to their rapid evolutionary rates compared to other proteins in humans. However, most of the studies on their accelerated evolution and selective forces behind it rely on DNA sequencing data. The objective of this study is to experimentally compare the function of one of these reproductive proteins in humans and our close evolutionary relatives to analyze the functional consequences of amino acid substitutions, which can then help us understand the possible factors contributing to its selection. To accomplish this aim, one of the most abundant seminal plasma proteins, kallikrein‐related peptidase 3 (KLK3) was used. KLK3 is also known as prostate‐specific antigen (PSA). Its physiological function is to liquefy the seminal coagulum, an important step for fertilization, releasing sperm cells for active motility. The formation of seminal coagulum is considered as a sperm competition strategy. Higher sperm competition was found to be correlated with more nonsynonymous substitutions in many seminal proteins. Therefore, their adaptive evolution is commonly attributed to sexual selection through post‐copulatory sperm competition. Previous studies showed that several codons from KLK3 orthologs have been evolving under positive selection and there is variation in dN/dS ratios among branches of primates, these were also confirmed by our multiple sequence alignment data. Based on the literature, the equivalent of the human KLK3 gene is found as chimeric KLK (cKLK, fusion of KLK2 and KLK3) in gorillas and gibbons. However, it has not yet been clarified whether the cKLK is enzymatically active or not. Considering the role of KLK3 in dissolution of seminal coagulum, its chimeric form might be related to the mating systems of gorillas and gibbons, who likely experience very low sperm competition. We hypothesize that amino acid substitutions have caused changes in KLK3 enzymatic activity across species with varying levels of sperm competition. To test this hypothesis, coding DNA sequences of KLK3/cKLK from human, chimpanzee, their hypothetical ancestor, gorilla, gibbon, and macaque were cloned into a mammalian expression vector with a C‐terminal His6‐tag. All these recombinant proteins were successfully expressed in HEK‐293T cells and purified using Nickel columns. After purification, they were treated with thermolysin to cleave the activation sequence from the core domain of KLK3 and subsequently analyzed for the protease activity on a synthetic fluorescent PSA substrate. Varying levels of enzyme activity were observed between species, supporting our hypothesis such that even a few substitutions were sufficient to cause changes in enzymatic activity. Enzyme kinetics (Kcat/Km) were also calculated for each species’ KLK3 protease activity. However, our preliminary data did not reveal a consistent trend of enzymatic activity in species with similar levels of sperm competition. This suggests that other potential selective pressures such as pathogen response and coevolution with its substrate could also be responsible for KLK3 adaptation along with sexual selection.