Hyaluronidases: Their Genomics, Structures, and Mechanisms of Action

Chem. Rev. 2006, 106, 818−839 818 Hyaluronidases: Their Genomics, Structures, and Mechanisms of Action Robert Stern† and Mark J. Jedrzejas*,‡ Children’s Hospital Oakland Research Institute, Oakland, California 94609, and Department of Pathology and UCSF Comprehensive Cancer Center, School of Medicine, University of California, San Francisco, California 94143-0511 Received April 26, 2005 Contents 8. Bacterial Hyaluronidases: Their Structure and 833 Mechanism of Action 1. Introduction 818 8.1. Polymeric Glycan-Binding Cleft: A Common 833 1.1. Overview of the Hyaluronidases 818 Feature of the Hyaluronidases and Other 1.2. Assays for Hyaluronidase Activity 820 Glycan-Degrading Enzymes 1.3. Eukaryote Hydrolase-Type of Hyaluronidases 820 8.2. Domain Structure Bacterial Hyaluronidases 834 1.4. Prokaryote Eliminase-Type of Hyaluronidases 820 8.3. Mechanism of Hyaluronan Degradation by 834 1.5. endo-â-Glucuronidase-Type of 820 Bacterial Hyaluronan Lyases Hyaluronidases 8.4. Streptomyces hyalurolyticus Hyaluronan 836 1.6. Fungal Hyaluronidases 820 Lyase 2. Hyaluronan as a Substrate for Catalysis 820 9. Summary 836 3. Properties of the Hyaluronidases 821 9.1. Conclusions 836 3.1. Designation of Hyaluronidases as a Class 821 9.2. Future Directions 836 within the Glycosidase Families of Enzymes 10. Abbreviations 837 3.2. The Term "Hyaluronidase" Is a Misnomer 822 11. Acknowledgments 837 3.3. Evolution of Hyaluronidases Can Be 822 12. Supporting Information 837 Postulated from Their Substrate Specificity 13. References 837 3.4. Digestion Products of Human and Bacterial 823 Hyaluronidase Catalysis 4. Identification of Mechanism of Hyaluronan 823 1. Introduction Degradation by Vertebrate-like Hyaluronidases 4.1. Catalytic Mechanism and Its Importance 823 1.1. Overview of the Hyaluronidases 4.2. Three-Dimensional Structures of the Bee 824 The hyaluronidases (Hyals) are classes of enzymes that Venom and Bovine PH-20 Hyaluronidases Suggest a Possible Mechanism of Action degrade, predominantly, hyaluronan (HA). The term “hyaluronidase” is somewhat of a misnomer since they have the 4.3. Identity of the Nucleophile and the Catalytic 824 Acid in the Active Site limited ability to degrade chondroitin (Ch) and chondroitin sulfates (ChS), albeit at a slower rate. It is a common 5. Human Hyaluronidases 825 misconception that the bacterial Hyals have absolute speci- 5.1. Properties of Human Hyaluronidases 825 ficity for HA. This is incorrect. Both bacterial1 and vertebrate 5.2. Considerations at the Genomic Level 827 enzymes degrade Ch and ChS. The plausible reason for this 6. Sequence Analysis and Homology and ab Initio 828 broader specificity is that chondroitins preceded HA in Three-Dimensional Modeling for Human evolution. For example, the nematode, Caenorhabditis el- Hyaluronidases egans, contains only Ch and no HA, with only one Hyal- 6.1. Fold Recognition Studies 828 like sequence (unpublished observations). This is most likely 6.2. Homology and ab Initio Modeling of the 828 a chondroitinase. It is plausible, therefore, that the vertebrate Human Hyaluronidases’ Structures Hyals evolved originally from pre-existing chondroitinases.1 7. Structures and Mechanism of Action of Human 829 This may explain why Hyals, recognizing their ancestral Hyaluronidases substrate, retain limited ability to also degrade Ch and ChS. 7.1. Description of the Model Structures 829 The Hyals from bacteria have been well characterized, and 7.2. Structure of the Active Site 829 much information is available (for representative publica- 7.3. Catalytic Mechanism of Hyaluronidase Activity 832 tions, see refs 2-5). The Hyals in vertebrate tissues, on the 7.4. Endolytic, Random Cut Pattern of 833 other hand, have not been studied extensively, due to the Hyaluronidase Activity lack of structural information. Such studies were more 7.5. Putative Functions of the C-Terminal 833 difficult and, therefore, more limited. In addition, vertebrate Domains Hyals are present at exceedingly low concentrations. In human serum, e.g., Hyal-1 is present at 60 ng/mL.6 They * To whom correspondence should be addressed. Phone: +1 510-450- have high specific activities that are unstable during the 7932. Fax: +1 510-450-7914. E-mail: [email protected]. Web: course of purification, requiring the constant presence of www.chori.org/investigators/jedrzejas.html. † University of California, San Francisco. detergents and protease inhibitors for their isolation. Many ‡ Children’s Hospital Oakland Research Institute. of such difficulties have been overcome, and a great deal of 10.1021/cr050247k CCC: $59.00 © 2006 American Chemical Society Published on Web 02/15/2006 Hyaluronidases: Genomics, Structures, Mechanisms Chemical Reviews, 2006, Vol. 106, No. 3 819 information is now available, facilitated in part by the Human Genome Project.7 Six Hyal sequences occur in the human genome, constitut- ing a newly recognized family of enzymes. They have similar catalytic mechanisms that contrast markedly with those of the bacterial Hyals. There is growing interest in these enzymes, as their HA substrate is achieving much attention. An outstanding review of the hyaluronidases was published 50 years ago by Karl Meyer, who was also the first to describe the chemical structure of HA.8 Interestingly, a chapter on mucopolysaccharidases, the former name for the hyaluronidases, was included in Volume 1 of Methods in Enzymology.9 The most recent overview of all of the Hyals appeared in 1971.10 Since that time, no comprehensive review has appeared. Robert Stern left Germany in 1938 for Seattle, Washington. He graduated from Harvard College in 1957 and obtained his M.D. degree from the Karl Meyer classified the Hyals into three distinct classes University of Washington (Seattle) in 1962. While a medical student, he of enzymes,8 based entirely on the biochemical analyses worked in the laboratories of Drs. Krebs and Fisher, who became Nobel available at the time. With the advent of sequence and laureates. He received his resident training in pathology at the NCI and structural data, we can now appreciate how remarkably was a research scientist at the NIH for 10 years. Since 1977, he has accurate Karl Meyer’s classification scheme was. No modi- been a member of the Pathology Department at the University of California, San Francisco. He is a board-certified Anatomic Pathologist, participating fication of his formulation is necessary. There are three major in the research, teaching, administrative, and diagnostic activities of the groups of Hyals, based on their mechanisms of action. Two Department. He directed the Ph.D. program in Experimental Pathology of the groups are endo-â-N-acetyl-hexosaminidases. One for 10 years. For the past decade, his research has focused on hyaluronan group includes the vertebrate enzymes that utilize substrate and the hyaluronidases, an outgrowth of an interest in malignancies of hydrolysis.11,12 The second group, which is predominantly connective tissue, stromal−epithelial interactions in cancer, and the biology bacterial, includes the eliminases that function by â-elimina- of the tumor extracellular matrix. His laboratory was the first to identify tion of the glycosidic linkage with introduction of an the family of six hyaluronidase sequences in the human genome. These 2-4,13-17 enzymes were then sequenced, expressed, and characterized. Subsequent unsaturated bond. As these enzymes catalyze the work has identified a catabolic pathway for hyaluronan. Dr. Stern was a breaking of a chemical bond by means other than hydrolysis Fulbright scholar in Germany (1984−5, He is currently a member of the or oxidation, and with the formation of a new double bond, editorial boards of Matrix Biology and the University of California Press. they are also termed lyases. Both terms, eliminase (or â-eliminase) and lyase, are used in the review interchange- ably. The third group are the endo-â-glucuronidases. These are found in leeches, which are annelids,18 and in certain crustaceans.19 No sequence data are available, and little is known about this potentially interesting class of enzymes. However, their mechanism of action resembles that of the eukaryotic or vertebrate enzymes more closely than that of the bacterial enzymes. Sequence data for vertebrate Hyals now provide op- portunities to formulate structure-function relationships, to examine probable mechanisms of catalysis, to identify putative substrate binding sites, and to consider the additional nonenzymatic functions of this family of multifunctional enzymes for two of the three groups, for the hydrolase and lyase types of Hyals, respectively.2 Such a review is Mark J. Jedrzejas received his B.A./M.S. in Physics from Jagellonian presented here, documenting some of the common and some University, Krakow, Poland, in 1988, an M. S. in Chemistry in 1992 from of the unusual features that distinguish each of these families Cleveland State University, and a Ph.D. in Structural Chemistry from of enzymes. Cleveland State University/Cleveland Clinic Foundation in 1993. He was appointed an Assistant Professor of Microbiology at the University of The primary objective of this review is to clarify what is Alabama at Birmingham in 1995. In 2001, Dr. Jedrzejas moved to the known about the structure and mode of action of all the Children’s Hospital Oakland Research Institute, as an Associate Scientist, Hyals. Since so little is known of the leech-type of Hyals, where he directs a structural biology group and continues his studies of the â-endoglucuronidases, the emphasis

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