The physiology and biochemistry of the fertility enzyme Proprotein Convertase SubtilisinKexin type 4

Abstract

This three-manuscript thesis focuses on the physiology and biochemistry of the fertility enzyme, Proprotein Convertase Subtilisin-Kexin type 4 (PCSK4). Efforts to develop effective, safe, and reversible methods of non-hormonal contraception especially for men have focussed either on sperm surface proteins involved in the functional maturation of sperm or on those responsible for sperm-egg interactions. PCSK4 is one of such proteins. It is a member of a family of serine endoproteinases involved in the proteolytic maturation of a wide array of inactive precursor proteins to their bioactive forms. It is primarily expressed in the gonads, and inactivation of its gene in mice causes male infertility and female subfertility. The first paper dealt with the reproductive function of PCSK4 by studying its subcellular localisation in testicular epithelium and on intact sperm, as well as its relevance for sperm acquisition of fertilizing ability. PCSK4 was detected in the acrosomal granules of round spermatids, in the acrosomal ridges of elongated spermatids, and on the sperm plasma membrane overlying the acrosome. Sperm from PCSK4-null mice underwent capacitation at a faster rate, they were induced to acrosome-react by lower concentrations of zona pellucida, and possessed egg-binding ability which was only half that of wildtype sperm. The second paper explored the molecular oasis of the ability of PCSK4-null sperm to undergo capacitation at a much faster rate than WT sperm, as well as their reduced egg-binding ability. It focussed primarily on sperm protein tyrosine phosphorylation and the proteolytic processing of the sperm-egg ligands ADAM2 and ADAM3. During sperm capacitation, proteins undergo more tyrosine phosphorylation and more ADAM2 proteolytic processing in PCSK4-null sperm than in WT sperm. Thus, alterations in signal transduction and proteolytic processing during capacitation may underlie the fertilisation incompetence of PCSK4-null sperm. The third paper investigated the biosynthesis, maturation, and transport of PCSK4. Mouse PCSK4 was identified to be tightly bound through hydrophobic interactions to water-insoluble or detergent-soluble components of the plasma membrane overlying the acrosome. Also human proPCSK4, cloned from human embryonic kidney cells (HEK293), is slightly converted into its active mature form, and it is probably retained inside the ER where it associates with Glucose-regulated protein 78/Imrnunogen Binding Protein (GRP78/BiP).