Hyaluronidase and its substrate hyaluronan: biochemistry, biological activities and therapeutic uses
Section snippets
Chemical and enzymatic nature of hyaluronidase
The term hyaluronidase was introduced to denote enzymes which degrade hyaluronate (HA). There are three main groups of enzymes with this specificity, but with different reaction mechanisms [1], i.e. (i) testicular-type hyaluronidase (hyaluronoglucosaminidase; hyaluronate 4-glycanohydrolase, EC 3.2.1.35), (ii) leech hyaluronidase (hyaluronate glycanohydrolase, EC 3.2.1.36) and (iii) bacterial hyaluronidase (hyaluronate lyase, EC 4.2.99.1).
Group (i) enzymes, which are the main topic of this
Pharmacodynamical and pharmacokinetic aspects of hyaluronidase
Hyaluronidase renders the tissue more readily permeable to injected fluids (spreading effect). The speed of absorption of drugs is increased and the discomfort due to subcutaneous or intramuscular injection is diminished. Capillary permeability is enhanced, as unequivocally demonstrated in rat skin after an i.c. dosage of hyaluronidase. The responsible permeability factor, a contaminating component, is not inhibited by serum factors [5]. Hyaluronidase enhances the therapeutic effect of
Measurement of hyaluronidase activity
With HA as substrate, all hyaluronidases can be determined by viscosimetric or turbidimetric methods [1]. Another method is the assay of reducing N-acetylglucosamine residues liberated from potassium hyaluronate [12]. Recently, a microtiter-based assay for hyaluronidase activity has been published [13], which may be used as a routine clinical laboratory procedure. Briefly, the free carboxyl groups of HA are biotinylated in a one-step reaction using biotin-hydrazide. This substrate is then
Physiologic role of hyaluronidase
HA fragmentation by hyaluronidase stimulates angiogenesis. Fibrotic healing of adult and late gestational wounds correlates with increased hyaluronidase activity and removal of HA [16]. In general, hyaluronidase is thought to play a role in HA homeostasis and metabolism, e.g. in the turnover of anterior chamber HA, and in the degradation of highly concentrated HA used as viscoelastic [17].
Hyaluronidase in tumors and cultivated cancer cell lines
Hyaluronidase levels were significantly increased in breast tumor metastases as compared to primary tumors. Three bands of activity were detected by zymographic analysis, corresponding to 49, 53 and 68 kDa, respectively. The same pattern was observed for cellular extracts of breast cancer cell line CAL51, demonstrating that hyaluronidase detected in tumor extracts has mainly a cellular origin [18]. Cell lines derived from primary tumors or from metastases produced hyaluronidase in culture. The
Hyaluronic acid, the substrate of hyaluronidase
HA is an alternating copolymer of varying chain length composed of repeating disaccharide units of the structure [→3)GlcNAc(β1→4)GlcUA(β1→], where GlcNAc is N-acetyl-d-glucosamine, GlcUA is d-glucuronic acid and the arrows and numbers, respectively, refer to the glycosidic linkage regions between monomeric moieties forming the unbranched heteropolysaccharide chain.
HA is widely distributed among the organs and tissues of the body and is found in such diverse locations as synovial fluid, the
Digestion of HA by hyaluronidase
Digestion with testicular hyaluronidase results in the exclusive cleavage of the N-acetylhexosaminidic linkages, the products comprising a series of oligosaccharides with N-acetylglucosamine at the reducing terminus. Upon exhaustive digestion, the tetrasaccharide is the major product, closely followed by the hexasaccharide and smaller amounts of higher oligosaccharides, while only a small proportion of disaccharide is found in a digest of this type. Chondroitin-6-sulfate is a much poorer
Cell adhesion to hyaluronate
HA is an abundant component of the extracellular matrix and is believed to be crucial in tissue remodeling, inflammation and tumorigenesis. Although several connective tissue proteins associate with HA, cells can also bind to HA directly via cell-surface HA-binding proteins. HA binds with high affinity (Kd=1–2×10−9 M) to the surface of cells due to the interaction of a single molecule of HA with multiple receptor sites. Binding of this type has been shown to occur in several types of cell lines
HA, CD44 and extravasation
The localization of circulating leukocytes within inflamed tissues occurs as the result of interactions with and migration across vascular endothelium and is governed in part by the expression of adhesion molecules on both cell types. Recently, a novel primary adhesion interaction (rolling) between structurally activated CD44 on lymphocytes and its major ligand HA on endothelial cells has been described, adding a new inducible endothelial adhesive molecule to the selectin and immunoglobulin
Cell activation by HA
Recent studies have shown that effector functions can be induced by HA through activation via CD44 in T cells, macrophages and B cells [34]. Purified B cells, but not T cells, from mice were found to show strong proliferative responses to soluble HA and not chondroitin sulfate. In vivo, splenic B cell proliferation could be induced by intraperitoneal injection of HA. A HA-specific regulation of cytokine production in human uterine fibroblasts has been described [35]. In addition, HA and IL-1β
HA binding in hematopoietic cells and tumor cells
Although most hematopoietic cells bear CD44, they rarely utilize it for recognition of the ligand HA [36]. Thus, only myeloma cells, and not normal hematopoietic cells, adhere to stromal cells through HA [37]. Cell adhesion molecules work in a carefully coordinated and cooperative fashion. Their activity can be controlled by expression or modulated after display on the cell surface. One way to regulate adhesion via HA is by growth factors, such as epidermal growth factor that stimulates cell
Cell surface HA
A thick HA coat is present around many types of cells in culture [40]. It is removed by Streptomyces hyaluronidase, suggesting that HA is the major structural component of the coat, which also contains fibronectin and proteoglycans. In particular, embryonic mesenchymal cells are rich in surface HA but poor in receptors. Cytotoxic interactions of lymphocytes with synovial fibroblasts and tumor cells are inhibited by the presence of HA-containing coats around target cells [40]. In both cases,
Cell aggregation via HA and CD44
Depending on HA receptor occupation, aggregation of lymphoma cells and macrophages is induced by the addition of HA. Endogenous cell surface HA causes cell aggregation by binding of HA on one cell to free receptor sites on an adjacent cell. The addition of a large excess of HA or the degradation of cell surface-associated HA by hyaluronidase both inhibit this aggregation. In vivo, embryonic cells are prevented from adhering to one another, an effect that might facilitate movement and prevent
Role of HA and hyaluronidase in morphogenesis
Studies of HA metabolism at discrete stages of early tissue development undergoing morphogenetic events have revealed close correlation between synthesis of HA and cell movement or proliferation as well as between removal of HA (by hyaluronidase) and differentiation. A common pattern in morphogenesis is accumulation of HA along the pathways of migration of mesenchymal cells, followed by its hyaluronidase-mediated removal and replacement by a sulfated proteoglycan-rich structural matrix during
Therapeutic uses of hyaluronidase and HA
Hyaluronidase and HA are widely used therapeutically in many fields, e.g. in ophthalmology, surgery, gynecology, etc. [46]. Due to the restricted space, we can only give a very short description of these therapeutical aspects.
During surgical interventions in the eye, HA is often injected intraoperationally to keep the anterior eye chamber intact or to protect the corneal endothelium during lens transplantation. The concomitant increase in intraocular pressure can be efficiently counteracted by
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