In time, the structural identity of testicular hyaluronidase was eventually elucidated, albeit through a rather circuitous
pathway. The purification to homogeneity of a hyaluronidase from bee venom, and the subsequent amino-acid sequencing and cDNA
cloning, revealed significant homology to the cDNA of a recently identified sperm-surface antigen, PH-20 (31, 32). Without
any knowledge of its hyaluronidase activity, the PH-20 protein had been previously identified as a glycosyl-phosphatidylinositol
(GPI) lipid-anchored antigen on the plasma membrane of sperm. Within this work, expression of PH-20 in rabbit kidney cells
showed the hyaluronidase activity associated with the membrane. Biochemical purification and molecular studies have identified
a family of hyaluronidase-like genes in the human genome (33). PH-20, however, is the predominant hyaluronidase in mammalian
testes, and among the hyaluronidase-like genes, only the PH-20 enzyme is active at neutral pH that degrades GAGs under physiologic
conditions. The human enzyme is a 509-amino acid glycoprotein anchored to the plasma membrane through a GPI moiety (34).
An rDNA-derived human PH-20 hyaluronidase enzyme
Hyaluronidase enzymes from testes had been commercially used as "spreading factors" for more than 50 years despite drawbacks
and inadequacies. The discovery of the human PH20 gene and its identification as a hyaluronidase, however, unlocked the potential
of producing a reliable recombinant human hyaluronidase enzyme.
Molecular engineering and manufacturing.
To develop a human PH-20 hyaluronidase, several issues had to be overcome, including the specific identification of the membrane
anchor and controversy over whether the soluble human protein retained enzymatic activity at physiologic pH (35, 36).Previous
attempts to produce the 382-amino acid bee venom enzyme in bacterial host systems showed only weak activity. Because the human
PH20 contained eight additional cysteine residues (12 total) with multiple N-linked glycosylation sites, expression in mammalian systems were considered for deletion mutagenesis (32). Earlier studies
showed that deletion mutagenesis of the carboxy terminus of the PH-20 gene near the proposed GPI anchor site or truncated
further to the equivalent carboxy terminus of the bee-venom hyaluronidase failed to produce an active enzyme in mammalian
cells (35). Therefore, a series of deletion mutants of the PH-20 cDNA were produced containing HIS6 epitope tags at the carboxyl
terminus. Soluble hyaluronidase activity was demonstrated in deletion mutants terminating after amino acids 477– 483. In contrast,
less than 10% activity was maintained when constructs terminated after amino acid 467 or when using the full-length PH-20
cDNA (37).
The soluble domain of the human PH-20 hyaluronidase cDNA (encoding amino acids 1–482) was cloned into a well-characterized
mammalian cell line (Chinese hamster ovary cells) in order to develop a recombinant human hyaluronidase (rHuPH20) for pharmaceutical
use. Purified to homogeneity, the rHuPH20 protein is a 61–63 kDa glycosylated enzyme with a specific activity 50–100 fold
greater than commercially available animal hyaluronidase extracts (on a unit/mg protein basis) (38). rHuPH20 exists as a homogeneous
preparation, in contrast to the multiple impurities in the commercial bovine-derived material. Indeed, only one minor 75 kDa
band in the smear of protein cross-reacted strongly with the anti-ram PH-20 monoclonal antibody, confirming that the hyaluronidase
enzyme is a minor component of animal-derived extracts, consistent with the low specific activity.
Comparison with bovine-derived material
It was important to compare the spreading activity of rHuPH20 to the original bovine preparations. It was possible that any
of the biologically active contaminants in the animal-derived material (including known contaminants such as proteinases,
anticoagulants, growth factors, vasopermeability factors, and immunoglobulins) could potentially contribute to the spreading
activity (39–43). These non-hyaluronidase impurities, however, did not contribute significantly. The spreading activity from
5 units of rHuPH20 (100,000 USP units/mg protein) was indistinguishable from that of 5 units of a crude commercial bovine
hyaluronidase preparation (approximately 700 units/mg protein) on a unit-per-unit basis when calibrated against the HA substrate
in vitro.
|
