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...

Sialic acids are important sugars at the reducing end of glycoproteins and glycolipids. They are among many other functions involved in cell-cell interactions, host-pathogen recognition and the regulation of serum half-life of glycoproteins. An important modification of sialic acids is O-acetylation, which can alter or mask the biological properties of the parent sialic acid molecule. The nature of mammalian sialate-O-acetyltransferases (EC 2.3.1.45) involved in their biosynthesis is still unknown. We have identified the human CasD1 (capsule structure1 domain containing 1) gene as a candidate to encode the elusive enzyme. The human CasD1 gene encodes a protein with a serine-glycine-asparagine-histidine hydrolase domain and a hydrophobic transmembrane domain. Expression of the Cas1 protein tagged with enhanced green fluorescent protein in mammalian and insect cells directed the protein to the medial and trans-cisternae of the Golgi. Overexpression of the Cas1 protein in combination with α-N-acetyl-neuraminide α-2,8-sialyltransferase 1 (GD3 synthase) resulted in an up to 40% increased biosynthesis of 7-O-acetylated ganglioside GD3. By quantitative real-time polymerase chain reaction, we found up to 5-fold increase in CasD1 mRNA in tumor cells overexpressing O-Ac-GD3. CasD1-specific small interfering RNA reduced O-acetylation in tumor cells. These results suggest that the human Cas1 protein is directly involved in O-acetylation of α2-8-linked sialic acids.

GD3 (CD60a) and its 9-O-acetylated variant (CD60b) are intracellular regulators of apoptosis in T lymphocytes. Surface expressed 9-O-acetyl- and 7-O-acetyl-GD3 (CD60b and CD60c) may have a functional impact on activated T and B cells. In order to investigate the balance between surface and intracellular expression and synthesis and degradation of these glycosphingolipids in human lymphocytes of various differentiation stages, we analyzed (i) expression of GD3 molecules on native T and B cells and thymocytes by flow cytometry and (ii) activity and regulation of possible key enzymes for CD60a,b,c synthesis and degradation at the transcriptional level. Both, surface and cytoplasmic expression of CD60a and CD60c was highest in tonsillar T cells. In thymocytes, CD60c outweighs the other CD60 variants and was mainly found in the cytoplasm. All lymphocyte preparations contained sialate O-acetyltransferase activity producing 7-O-acetyl-GD3. Sialidase activity was highest in peripheral blood lymphocytes followed by thymocytes and tonsillar T and B cells. Transcription of GD3 synthase (ST8SiaI), the key enzyme for GD3 synthesis, was highest in tonsillar T cells, whereas transcriptional levels of sialidase NEU3 and O-acetylesterase H-Lse were lowest in activated T cells. This balance between enzymes of sialic acid metabolism may explain the strong overall staining intensity for all GD3 forms in T cells. Both CASD1, presumably encoding a sialic acid-specific O-acetyltransferase, and H-Lse showed highest transcription in peripheral B lymphocytes corresponding to the low expression of CD60b and c in these cells. Our data point to regulatory functions of these anabolic and catabolic key enzymes for the expression of GD3 and its O-acetylated variants in lymphocytes at a given differentiation stage.