Chemistry and Biology of Hyaluronan

Hari G. Garg, Charles A. Hales

• Publisher: Elsevier Science
• Year: 2004
• ISBN:
  9780080472225
• Topic: Biological Sciences
• Subtopic: Biochemistry
• Index: Biological Sciences

It was probably the French chemist Portes, who first reported in 1880 that the mucin in the vitreous body, which he named hyalomucine, behaved differently from other mucoids in cornea and cartilage. Fifty four years later Karl Meyer isolated a new polysaccharide from the vitreous, which he named hyaluronic acid. Today its official name is hyaluronan, and modern-day research on this polysaccharide continues to grow.

Expertly written by leading scientists in the field, this book provides readers with a broad, yet detailed review of the chemistry of hyaluronan, and the role it plays in human biology and pathology. Twenty-seven chapters present a sequence leading from the chemistry and biochemistry of hyaluronan, followed by its role in various pathological conditions, to modified hylauronans as potential therapeutic agents and finally to the functional, structural and biological properties of hyaluronidases. Chemistry and Biology of Hyaluronan covers the many interesting facets of this fascinating molecule, and all chapters are intended to reach the wider research community.

• Comprehensive look at the chemistry and biology of hyaluronans
• Essential to Chemists, Biochemists and Medical researchers
• Broad yet detailed review of this rapidly growing research area

Chapter 1 Solution Properties of Hyaluronan

TIM. HARDINGHAM

Wellcome Trust Centre for Cell-Matrix Research, School of Biological Sciences, University of Manchester, Manchester, UK

I. Introduction

Hyaluronan (HA) is a high molecular weight (10⁵ – 10⁷ Da) unbranched glycosaminoglycan, composed of repeating disaccharides (β1-3 D-N-acetylglucosamine, β1-4 D-glucuronic acid). It is a widely distributed component of the extracellular matrix of vertebrate tissues (1). It acts as a scaffold for the binding of other matrix molecules including aggrecan and other members of the hyalectan family (2,3). It has an interesting mechanism of synthesis in which chain extension is by monosaccharide addition at the reducing end of the chain (4). This is thus in the opposite direction to other vertebrate glycosaminoglycans. It also appears to be synthesised by a glycosyltransfererase with two catalytic activities, for the glucuronic acid transfer and the N-acetylglucosamine transfer (5). The enzyme also appears to be embedded in the plasma membrane of cells with the product being translocated out of the cell as synthesis proceeds and there are three related mammalian HA synthases.

II. Historical Perspective

HA was initially discovered and named hyaluronic acid in a paper published in 1934 by Karl Meyer (6). It was isolated from vitreous of the eye as a polysaccharide containing D-glucuronic acid and D-N-acetylglucosamine, but it was not until 20 years later that he completed the determination of its structure and showed that it contained a repeating β1-3, β1-4 linked disaccharide. In the meantime, HA was isolated from many tissue sources, including synovial fluid, cock’s comb and umbilical cord. Its extraction from tissues was not easy and HA preparations always retained some protein. The controversy over whether HA was linked to a protein remained in the literature for many years (see Refs. 7–9) and the issue was particularly debated during the 1960s and 1970s as other structurally related glycosaminoglycans were characterised and their covalent attachment to protein as proteoglycans was being explored. The question naturally arose, was HA a proteoglycan? The issue was not easily resolved because of the unusual properties of HA and the difficulty of preparing it free of protein using classical biochemical methods. Exhaustive isolation and fractionation methods resulted in a low but significant content of residual protein (∼0.5% w/w). This combined with the high molecular weight of HA left the possibility that each chain was attached to a small protein. At the same time, important discoveries were establishing highly specific interactions of HA with proteins, the first of which identified its role in binding to aggrecan and forming supramolecular aggregates (10). However, the issue of HA’s covalent link to protein as a requirement for biosynthesis was finally resolved with the discovery of the mechanism of biosynthesis of HA (4) and the subsequent cloning of the HA synthase enzyme (5), which showed that HA could be made without any protein primer. It is interesting that subsequently novel mechanisms have been discovered by which covalent protein–HA bonds can be formed extracellularly with inter α-trypsin inhibitor and related proteins (11) and this may explain some of the difficulty of removing final traces of protein from HA prepared from some tissue sources. It is now clear that HA is synthesised by the cells of higher organisms without the need for any protein primer. HA has thus evolved from quite a different evolutionary origin from the other structurally related glycosaminoglycans, which are synthesised attached to proteins and whose chains are extended by single sugar addition to the non-reducing end of each chain.

From its initial isolation, the physical properties of HA have been the dominant feature that distinguished it from other components of extracellular matrix. In the early work characterising HA, even including the simple determination of its molecular weight presented great difficulty. The properties of HA provided a challenge to the classical biophysical methods, in which simple analysis was developed for proteins and required that the properties approached those of perfect Newtonian solutes. The behaviour of HA in solutions even at low concentration is far from Newtonian or ‘ideal’, and it presented a challenge in the 1950s and 1960s that some very distinguished researchers took up, notably Sandy Ogston, Torvard Laurent, Endre (Bandi) Balazs and later Bob Cleland (7,8,12–17). Their work established a theoretical and experimental framework that underpins HA research to this day and the concepts they developed are fundamental to understanding the biophysical properties of HA.

The key elements they identified were:

• It was a high molecular weight unbranched polysaccharide, which behaved as a stiffened random coil in solution (Fig. 1).

• It occupied a large hydrated volume and therefore showed solute–solute interactions at unusually low concentration.

• It showed excluded volume effects, as it restricted access to this domain by other macromolecules.

• These properties were compounded by the fact that HA was a polyelectrolyte and therefore the solution properties were also greatly affected by ionic strength.

• HA was also established to be polydisperse and its properties were therefore the aggregate properties of a population of molecules of varying chain length, rather than those of a unique species.

Figure 1 Models of hyaluronan behaviour in solution. In dilute solution, hyaluronan behaves as a stiffened random coil. The presence of linked segments would act in opposition to chain stiffening in determining the hydrated domain. In concentrated solutions, stiffened random coils show entanglement; they form viscoelastic solutions and retain flow and do not become gels. The presence of linked segments would create a network and lead to gel formation.

Much of this early work focussed on relating biophysical measurements of light scattering, osmometry, viscosity and sedimentation to models of behaviour. An important early development in this process was the recognition of the need to extrapolate experimental results to vanishingly low concentrations in order to determine intrinsic properties and free the measured parameters from non-ideal effects caused by interaction between adjacent molecules. With an evolving understanding of non-ideal behaviour of polyelectrolyte biopolymers such as HA it was found possible to obtain consistent results from different biophysical techniques.

There is a theme that runs through the fascinating history o...

Table of contents

• Cover image
• Title page
• Table of Contents
• Copyright
• Dedication
• Preface
• Contributors
• Chapter 1: Solution Properties of Hyaluronan
• Chapter 2: Methods for Analysis of Hyaluronan and Its Fragments
• Chapter 3: Methods for Determination of Hyaluronan Molecular Weight
• Chapter 4: Biodegradation of Hyaluronan
• Chapter 5: The Hyaluronan Receptor: CD44
• Chapter 6: The Role of the Hyaluronan Receptor RHAMM in Wound Repair and Tumorigenesis
• Chapter 7: Signal Transduction Associated with Hyaluronan
• Chapter 8: Structural and Functional Diversity of Hyaluronan-Binding Proteins
• Chapter 9: Biological Function of SHAP–Hyaluronan Covalent Complex
• Chapter 10: Hyaluronan and Associated Proteins in the Visual System
• Chapter 11: Hyaluronan in the Pulmonary Alveolus and Interstitium
• Chapter 12: Hyaluronan in Ventilator-Induced Lung Injury
• Chapter 13: The Role of Hyaluronan in Cancer
• Chapter 14: Hyaluronan in Atherosclerosis and Restenosis
• Chapter 15: Hyaluronan in the Airways
• Chapter 16: Hyaluronan Biology in Vocal Fold Morphology and Biomechanics
• Chapter 17: Hyaluronan in Aging
• Chapter 18: Hyaluronan and Scarring
• Chapter 19: Hyaluronan in the Epidermis and Other Epithelial Tissues
• Chapter 20: Viscoelastic Properties of Hyaluronan and Its Therapeutic Use
• Chapter 21: Medical Application of Hyaluronan
• Chapter 22: Therapeutic Biomaterials from Chemically Modified Hyaluronan
• Chapter 23: Medicinal Uses of Modified Hyaluronate
• Chapter 24: Hyaluronan in the Treatment of Ocular Surface Disorders
• Chapter 25: The Hyaluronan Synthases
• Chapter 26: Molecular Genetic Dissection of Hyaluronan Function in the Mouse
• Chapter 27: Functional, Structural and Biological Properties of Hyaluronidases
• Subject index