In Vitro Mutagenesis of PH‐20 Hyaluronidase from Human Sperm

Abstract

The cDNA encoding PH-20 hyaluronidase from human sperm has been mutated at five positions by in vitro mutagenesis. We have changed three acidic amino acids and two arginine residues that are conserved in the sequence of mammalian PH-20 polypeptides as well as in the hyaluronidases from bee and hornet venom. Of the former, the mutants [Gln113]PH-20 and [Gln249]PH-20 had no detectable enzymatic activity; the mutant [AsnllllPH-20 had about 3% activity. The mutant [Thr252]PH-20 was also inactive, while [Gly176]PH-20 had only about 1 % activity. This indicates that the PH-20 hyaluronidases, like numerous enzymes that hydrolyze glycosidic bonds, have acidic amino acids in their active site. Moreover, for the binding of the substrate, the polyanion hyaluronan, arginine residues appear to be essential.Keywords: PH-20 hyaluronidase; in vitro mutagenesis ; spermatozoa.Hyaluronan (hyaluronic acid) is a ubiquitous component of the extracellular matrix of vertebrates. This glycosaminoglycan, which is composed of alternating units of N-acetylglucosamine and glucuronic acid, can form highly viscous solutions and thereby influence the properties of this matrix. Hyaluronan has been implicated in many biological processes including fertilization, embryonic development, cell migration, and differentiation, wound healing, inflammation, and growth and metastasis of tumor cells (Laurent and Fraser, 1992). Three types of hyaluronidases are known which are widely distributed in nature (see Meyer, 1971;Kreil, 1995;Frost et al., 1996 for reviews). One group are endo-N-acetylhexosaminidases that hydrolyze hyaluronan with a molecular mass of over 1 X lo6 Da mostly to tetrasaccharides. These enzymes have been detected in diverse sources such as mammalian testes and insect venoms. A few years ago, the amino acid sequence of bee venom hyaluronidase was elucidated via cDNA cloning . The sequence of this enzyme was found to be homologous to PH-20, a polypeptide localized on the head of guinea pig sperm (Lathrop et al., 1990). It could subsequently be demonstrated that PH-20 from different mammalian species has hyaluronidase activity Lin et al., 1994a;Cherr et al., 1996).A comparison of the amino acid sequences of bee and hornet venom hyaluronidases Lu et al., 1995) and those of the human, monkey, mouse, and guinea pig PH-20 proteins (Lathrop et al., 1990; Lin et al., 1994a, b) demonstrated that in a common region encompassing about 340 amino acids, 57 amino acids are conserved in all of these proteins (Fig. 1). These include four cysteine residues forming two disulfide bridges. In addition, we assume that the residues essential for substrate binding and catalysis are also conserved between the hymenopteran and mammalian enzymes. At present, nothing is known about the active site of hyaluronidases. Based on a few assumptions, we have now addressed the question which amino acids in these enzymes are essential for the hydrolysis of hyaluronan. Binding of the acidic glycosaminoglycan to the enzyme probably involves ionic interaction with basic am...

Mouse testicular hyaluronidase-like proteins SPAM1 and HYAL5 but not HYALP1 degrade hyaluronan

“…However, major spatial differences were evident in the structure of HYALP1, in particular amino acid moieties that appear to be important for the proper positioning of the substrate with regard to the acidic amino acid moieties within the active centre ( Figure 6B). The active sites of these enzymes consist of aspartate and glutamate residues interspaced by one amino acid residue [45,46]. Notably, HYAL5 and SPAM1, yet also HYALP1, show this particular feature.…”

Structure of Human Hyaluronidase-1, a Hyaluronan Hydrolyzing Enzyme Involved in Tumor Growth and Angiogenesis,

“…Its side chain is partially buried, forming a hydrogen bond with Asn229 (located on R5) and a salt bridge with Arg271 (located on R6), both invariant residues in the human hyaluronidases. Replacement of this glutamic acid by a glutamine residue in hPH-20 led to loss of activity (41). The deleterious effect of a mutation in this position is expected to arise from reduced protein stability due to removal of the electrostatic interaction between R5 and R6.…”

HYAL1-v1, An Alternatively Spliced Variant of HYAL1 Hyaluronidase: A Negative Regulator of Bladder Cancer

“…We have previously reported that these 90 bases make up the entire exon 2, and therefore, HYAL1-v1 is generated by splicing of exon 2 (26). It is noteworthy that the 30-amino acid sequence encoded by exon 2 is not a part of the catalytic domain; however, it is well conserved in all six human hyaluronidases and also in the bee hyaluronidase (33)(34)(35)(36). The observation that HYAL1-v1 is enzymatically inactive and that a similar splice variant of HYAL-3 (HYAL3-v1) is also enzymatically inactive may explain why this 30-amino acid sequence that is encoded by a separate exon in both HYAL1 and HYAL3 is well conserved (26).…”