Individual cartilage aggrecan macromolecules and their constituent glycosaminoglycans visualized via atomic force microscopy (original) (raw)
Related papers
The Glycosaminoglycan Attachment Regions of Human Aggrecan
Journal of Biological Chemistry, 2006
Aggrecan possesses both chondroitin sulfate (CS) and keratan sulfate (KS) chains attached to its core protein, which reside mainly in the central region of the molecule termed the glycosaminoglycan-attachment region. This region is further subdivided into the KS-rich domain and two adjacent CS-rich domains (CS1 and CS2). The CS1 domain of the human is unique in exhibiting length polymorphism due to a variable number of tandem amino acid repeats. The focus of this work was to determine how length polymorphism affects the structure of the CS1 domain and whether CS and KS chains can coexist in the different glycosaminoglycan-attachment domains. The CS1 domain possesses several amino acid repeat sequences that divide it into three subdomains. Variation in repeat number may occur in any of these domains, with the consequence that CS1 domains of the same length may possess different amino acid sequences. There was no evidence to support the presence of KS in either the CS1 or the CS2 domains nor the presence of CS in the KS-rich domain. The structure of the CS chains was shown to vary between the CS1 and CS2 domains, particularly in the adult, with variation occurring in chain length and the sulfation of the non-reducing terminal N-acetyl galactosamine residue. CS chains in the adult CS2 domain were shorter than those in the CS1 domain and possessed disulfated terminal residues in addition to monosulfated residues. There was, however, no change in the sulfation pattern of the disaccharide repeats in the CS chains from the two domains.
Structure and function of aggrecan
Cell Research, 2002
Aggrecan is the major proteoglycan in the articular cartilage. This molecule is important in the proper functioning of articular cartilage because it provides a hydrated gel structure (via its interaction with hyaluronan and link protein) that endows the cartilage with load-bearing properties. It is also crucial in chondroskeletal morphogenesis during development. Aggrecan is a multimodular molecule expressed by chondrocytes. Its core protein is composed of three globular domains (G1, G2, and G3) and a large extended region (CS) between G2 and G3 for glycosaminoglycan chain attachment. G1 comprises the amino terminus of the core protein. This domain has the same structural motif as link protein. Functionally, the G1 domain interacts with hyaluronan acid and link protein, forming stable ternary complexes in the extracellular matrix. G2 is homologous to the tandem repeats of G1 and of link protein and is involved in product processing. G3 makes up the carboxyl terminus of the core protein. It enhances glycosaminoglycan modification and product secretion. Aggrecan plays an important role in mediating chondrocyte-chondrocyte and chondrocyte-matrix interactions through its ability to bind hyaluronan.
Probing interactions between aggrecan and mica surface by the atomic force microscopy
Journal of Polymer Science Part B: Polymer Physics, 2010
Aggrecan is a bottlebrush shaped macromolecule found in the extracellular matrix of cartilage. The negatively charged glycosaminoglycan (GAG) chains attached to its protein backbone give aggrecan molecules a high charge density, which is essential for exerting high osmotic swelling pressure and resisting compression under external load. In solution, aggrecan assemblies are insensitive to the presence of calcium ions, and show distinct osmotic pressure versus concentration regimes. The aim of this study is to investigate the effect of ionic environment on the structure of aggrecan molecules adsorbed onto well-controlled mica surfaces. The conformation of the aggrecan was visualized using Atomic Force Microscopy. On positively charged APS mica the GAG chains of the aggre-can molecules are distinguishable, and their average dimensions are practically unaffected by the presence of salt ions. With increasing aggrecan concentration they form clusters, and at higher concentrations they form a continuous monolayer of conforming molecules. On negatively charged mica, the extent of aggrecan adsorption varies with salt composition. Understanding aggrecan adsorption onto a charged surface provides insight into its interactions with bone and implant surfaces in the biological milieu.
Solution Structure and Dynamics of Cartilage Aggrecan
Biomacromolecules, 2006
We studied the structure and dynamics of porcine laryngeal aggrecan in solution using a range of noninvasive techniques: dynamic light scattering (DLS), small-angle neutron scattering (SANS), video particle tracking (VPT) microrheology, and diffusing wave spectroscopy (DWS). The data are analyzed within the framework of a combined static and dynamic scaling model, and evidence is found for reptation of the comb backbones with unentangled side-chain dynamics. Small-angle neutron scattering indicated standard polyelectrolyte scaling of the mesh size ( ) with concentration (c) in semidilute solutions for the whole aggrecan aggregate, ) Ac -0.47(0.04 , with the prefactor (A) implying there is on average 60 nm between the aggrecan subunits along the backbone. VPT demonstrated large exponents for the power law dependence of the intrinsic viscosity (η) on the polymer concentration in the semidilute concentration regime, η ∼ c R ; with R equal to 2.04 ( 0.06 and 1.95 ( 0.08 for the assembled and disassembled aggrecan aggregates, respectively. DWS at high frequencies (10 4 -10 5 Hz) gave evidence for internal Rouse modes of the aggrecan monomers, independent of the degree of self-assembly of the molecules. Figure 1. Schematic diagram of the process of self-assembly of aggrecan aggregate from monomer and hyaluronic acid.
Molecular cloning and analysis of the protein modules of aggrecans
Experientia, 1993
The large aggregating chondroitin sulfate proteoglycan of cartilage, aggrecan, has served as a prototype of proteoglycan structure. Molecular cloning has elucidated its primary structure and revealed both known and unknown domains. To date the complete structures of chicken, rat and human aggrecans have been deduced, while partial sequences have been reported for bovine aggrecan. A related proteoglycan, human versican, has also been cloned and sequenced. Both aggrecan and versican have two lectin domains, one at the amino-terminus which binds hyaluronic acid and one at the carboxyl-terminus whose physiological ligand is unknown. Both lectins have homologous counterparts in other types of proteins. Within the aggrecans the keratan sulfate domain may be variably present and also has a prominent repeat in some species. The chondroitin sulfate domain has three distinct regions which vary in their prominence in different species. The complex molecular structure of aggrecans is consistent with the concept of exon shuffling and aggrecans serve as suitable prototypes for comprehending the evolution of multi-domain proteins.
Journal of Biological Chemistry, 2001
Aggrecan is the major proteoglycan in the extracellular matrix of cartilage. A notable exception is nanomelic cartilage, which lacks aggrecan in its matrix. The example of nanomelia and other evidence leads us to believe that the G3 domain plays an important role in aggrecan processing, and it has indeed been confirmed that G3 allows glycosaminoglycan (GAG) chain attachment and product secretion. However, it is not clear how G3, which contains at least a carbohydrate recognition domain (CRD) and a complement binding protein (CBP) motif, plays these two functional roles. The present study was designed to dissect the mechanisms of this phenomenon and specially 1) to determine the effects of various cysteine residues in GAG modification and product secretion as well as 2) to investigate which of the two processing events is the critical step in the product processing. Our studies demonstrated that removal of the two amino-terminal cysteines in the CRD motif and the single cysteine in the amino terminus of CBP inhibited secretion of CRD and CBP. Use of the double mutant CRD construct also allowed us to observe a deviation from the usual strict coupling of GAG modification and product secretion steps. The presence of a small chondroitin sulfate fragment overcame the secretioninhibitory effects once the small chondroitin sulfate fragment was modified by GAG.
Proteoglycans are major constituents of the extracel-Aggrecan is a key component of the cartilage matrix. lular matrix of articular cartilage and are largely re-During aging, many changes occur in its composition sponsible for the high resistance to compression of this and structure; in particular, there is an increase in the load-bearing tissue. This resistance to compression is proportion of lower molecular weight monomers and mainly due to the high osmotic pressure of the nega-of the ''free'' binding region. An important question tively charged glycosaminoglycan (GAG) 2 chains. The has been whether these changes represent alterations proteoglycans (aggrecan) are organized in the matrix in biosynthesis or whether they are due to the accumu-through a highly specific interaction to form large ag-lation with age of the partially degraded fragments gregates consisting of a central filament of hyaluronan, of the originally synthesized large monomer. In the to which aggrecan monomers are attached via the hya-present work we have used an independent tool, viz., luronan-binding domain (G1) of their core proteins, the extent of racemization of aspartic acid to study the with the interaction being stabilized by the presence molecular ''age'' of different buoyant density fractions of a link protein. Aggregate formation is very important of the aggrecan of human articular cartilage, as well from the physiological point of view, since it ensures as of isolated free binding region and link protein. By the retention of aggrecan within the collagen network. measuring the D/L Asp ratio of the different aggrecan
Aggrecan, an unusual polyelectrolyte: Review of solution behavior and physiological implications
Acta Biomaterialia, 2012
Aggrecan is a high-molecular-weight, bottlebrush-shaped, negatively charged biopolymer that forms supermolecular complexes with hyaluronic acid. In the extracellular matrix of cartilage, aggrecan-hyaluronic acid complexes are interspersed in a collagen meshwork and provide the osmotic properties required to resist deswelling under compressive load. In this review we compile aggrecan solution behavior from different experimental techniques, and discuss them in the context of concentration regimes that were identified in osmotic pressure experiments. At low concentrations, aggrecan exhibits microgel-like behavior. With increasing concentration, the bottlebrushes self-assemble into large complexes. In the physiological concentration range (2 < c aggrecan < 8% w/w), the physical properties of the solution are dominated by repulsive electrostatic interactions between aggrecan complexes. We discuss the consequences of the bottlebrush architecture on the polyelectrolyte characteristics of the aggrecan molecule, and its implications for cartilage properties and function.