Designed human serum hyaluronidase 1 variant, HYAL1DeltaL, exhibits activity up to pH 5.9.

Hyaluronidases from diverse species and sources have different pH optima. Distinct mechanisms with regard to dynamic structural changes, which control hyaluronidase activity at varying pH, are unknown. Human serum hyaluronidase 1 (HYAL1) is active solely below pH 5.1. Here we report the design of a HYAL1 variant that degrades hyaluronan up to pH 5.9. Besides highly conserved residues in close proximity of the active site of most hyaluronidases, we identified a bulky loop formation located at the end of the substrate binding crevice of HYAL1 to be crucial for substrate hydrolysis. The stretch between cysteine residues 207 and 221, which normally contains 13 amino acids, could be replaced by a tetrapeptide sequence of alternating glycine serine residues, thereby yielding an active enzyme with an extended binding cleft. This variant exhibited hyaluronan degradation at elevated pH. This is indicative for appropriate substrate binding and proper positioning being decisively affected by sites far off from the active center.

Leptin receptor/CD295 is upregulated on primary human mesenchymal stem cells of advancing biological age and distinctly marks the subpopulation of dying cells.

During the lifetime of an adult organism, stem cells face extrinsic and intrinsic aging. Mesenchymal stem cells (MSC) can be expanded in culture, and the proliferation potential of individual cell isolates before growing senescent appear to be dependent on fitness and age of the donor, respectively. To date no molecular markers are available, which specifically reflect the degree of cellular aging in a population of MSC. Employing a genomic approach, we noticed that the gene encoding leptin receptor (also termed OB-R) is differentially regulated in MSC derived from aged donors as well as in MSC that had been stressed due to cultivation under hyperoxic conditions. We further observed that the leptin receptor transcript levels in primary MSC isolates are inversely correlated with the prospective number of generations that are ahead of these cells in culture, i.e., the number of population doublings that will occur in long term culture prior to cessation of growth due to replicative senescence. The MSC subpopulation, which exhibited distinctly elevated levels of leptin receptor or CD295 at the cell surface, is indistinguishable from dying cells. Considered together with the observation that primary MSC derived from healthy individuals showed proliferation capacities that declined at differentially increasing rates, we concluded that attenuation of MSC proliferation potential during aging greatly relies on the strictly increasing withdrawal of cells due to cell death.

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

Besides SPAM1 (sperm adhesion molecule 1; formerly named PH-20), further hyaluronidase-like proteins, HYAL5 (hyaluronoglucosaminidase 5) and HYALP1 (hyaluronoglucosaminidase pseudogene 1) are also expressed in murine testicular tissue. As they share a high degree of sequence similarity with known hyaluronidases, all three polypeptides could potentially exhibit hyaluronidase activity, a function that is beneficial for spermatozoa in order to penetrate the hyaluronan-rich cumulus, which surrounds the oocyte. Recently, it was reported that SPAM1-deficient mice are fertile and spermatozoa derived from mutant mice still exhibit hyaluronidase activity [Baba, Kashiwabara, Honda, Yamagata, Wu, Ikawa, Okabe and Baba (2002) J. Biol. Chem. 277, 30310-30314]. We have now recombinantly expressed mouse SPAM1, HYAL5 and HYALP1 in Xenopus laevis oocytes and determined their respective expression pattern in testis. Transcripts of all three genes are expressed in seminiferous tubules in regions where maturing spermatogenic cells reside. SPAM1 and HYAL5 but not HYALP1 proteins exhibit hyaluronidase activity at neutral pH. The two active hyaluronidases are both bound to the cell surface via a glycosylphosphatidylinositol anchor. Furthermore, structural characteristics are discussed that are necessary for hyaluronidases in order to exhibit hyaluronan cleavage.