Cartilage proteoglycan aggregates. Electronmicroscopic studies of native and fragmented molecules

Heinegård, Dick; Lohmander, Stefan; Thyberg, Johan

doi:10.1042/bj1750913

Cited by 47 publications

(16 citation statements)

References 23 publications

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“…The structure of the KS-rich region suggests a relatively extended and rigid conformation. This is supported by electron microscopy of spread proteoglycan aggregates after rotary shadowing, showing that the CS-rich region is clearly separated from the central filament of hyaluronan (6). It appears that the KS-rich region acts as a spacer within the proteoglycan aggregate.…”

supporting

confidence: 57%

Association of the Aggrecan Keratan Sulfate-rich Region with Collagen in Bovine Articular Cartilage

Hedlund¹,

Hedbom²,

Heinegård³

et al. 1999

Journal of Biological Chemistry

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Aggrecan, the predominant large proteoglycan of cartilage, is a multidomain macromolecule with each domain contributing specific functional properties. One of the domains contains the majority of the keratan sulfate (KS) chain substituents and a protein segment with a proline-rich hexapeptide repeat sequence. The function of this domain is unknown but the primary structure suggests a potential for binding to collagen fibrils. We have examined binding of aggrecan fragments encompassing the KS-rich region in a solid-phase assay. A moderate affinity (apparent K d ‫؍‬ 1.1 M) for isolated collagen II, as well as collagen I, was demonstrated. Enzymatic digestion of the KS chains did not alter the capacity of the peptide to bind to collagen, whereas cleavage of the protein core abolished the interaction. The distribution of the aggrecan KS-rich region in bovine tarsometatarsal joint cartilage was investigated using immunoelectron microscopy. Immunoreactivity was relatively low in the superficial zone and higher in the intermediate and deep zones of the uncalcified cartilage. Within the pericellular and territorial matrix compartments the epitopes representing the aggrecan KSrich region were detected preferentially near or at collagen fibrils. Along the fibrils, epitope reactivity was non-randomly distributed, showing preference for the gap region within the D-period. Our data suggest that collagen fibrils interact with the KS-rich regions of several aggrecan monomers aligned within a proteoglycan aggregate. The fibril could therefore serve as a backbone in at least some of the aggrecan complexes.Articular cartilage matrix can be regarded as a fiber-reinforced composite material (1), where aggrecan complexes are entangled within a network of collagen fibrils. The aggrecan complexes, constituting about 90% of the proteoglycan content (2), endow the matrix with high osmotic pressure, compressive stiffness, and resilience, whereas collagen is essential for the tensile strength of the tissue (3). Different mechanical properties of the composite depend on these major constituents and how they are assembled and stabilized by intermolecular interactions. The capacity of cartilage to withstand mechanical stress depends upon its structural integrity and, hence, numerous interactions between the matrix components. Indeed, the molecules are so tightly associated that most of the tissue constituents require denaturing solvents or proteases for extraction. This has hampered studies of molecular function. To gain insight into the physiology of articular cartilage, it is necessary to identify and characterize interactions between the matrix constituents, particularly those involving the collagen.In the present study we focused on a domain of the aggrecan molecule containing the majority of the keratan sulfate (KS) 1 chain substituents and a protein segment with a hexapeptide repeat sequence (4). The second globular domain (G2) is localized adjacent to this KS-rich region, on the N-terminal side. The first globular domain (G1), which r...

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supporting

confidence: 57%

Association of the Aggrecan Keratan Sulfate-rich Region with Collagen in Bovine Articular Cartilage

Hedlund¹,

Hedbom²,

Heinegård³

et al. 1999

Journal of Biological Chemistry

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“…The hyaluronic acid chain and individual proteoglycan were clearly distinguishable. Their length and width were proximate to studies using EMs (Buckwalter and Rosenberg 1982, Buckwalter et al 1984, Cornelissen and de Ridder 1993, Heinegard et al 1978, Morgelin et al 1988, Rosenberg et al 1975, Tang et al 1996, Thyberg 1977, Thyberg et al 1975. In addition to the dimension in the two-dimensional (2-D) plane, the height of the molecules could be accurately measured.…”

Section: Resultsmentioning

confidence: 99%

The structure of proteoglycan aggregate determined by atomic force microscopy

Yeh

Luo

2004

Scanning

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Summary: Proteoglycan aggregate is the major extracellular matrix component in cartilage, comprising about 18% of the dry weight of hyaline cartilage. The proteoglycan aggregate is the major substance in cartilage which resists compression in the joint. The purpose of this study was to utilize the newly developed imaging technique, Atomic force Microscopy (AFM), to visualize the ultrastructure of proteoglycan aggregates. The proteoglycan aggregate molecules were imaged in air using the tapping mode of the AFM. The images illustrated the ultrastructure of the aggregates, especially the individual proteoglycan and the core hyaluronic acid. In addition to the length and width of each molecule, the height of the proteoglycan aggregates and the individual proteoglycans could be directly measured. The images of the ultrastructures of proteoglycan aggregates visualized from the AFM are comparable with those using conventional electron microscopy approaches. Nevertheless, the sample preparation for AFM imaging does not involve fixation, staining, coating, and other routine procedures required for traditional electron microscopy imaging. Thus, this technique could be a simple alternative approach for future analysis of proteoglycan aggregate and its assembly.

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“…Stabilization of the aggrecan network in cartilage involves strong non-covalent interactions with link protein and hyaluronan at the N-terminal G1 globular domain and G3-mediated interactions at the C terminus that may be influenced by alternative splicing (77,78). Given the abundance of aggrecan and link protein in cartilage, it was not surprising that these two components generated the highest spectral count values in juvenile cartilage.…”

Section: Discussionmentioning

confidence: 99%

Comprehensive Profiling of Cartilage Extracellular Matrix Formation and Maturation Using Sequential Extraction and Label-free Quantitative Proteomics

Wilson

Diseberg

Gordon

et al. 2010

Molecular & Cellular Proteomics

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Articular cartilage is indispensable for joint function but has limited capacity for self-repair. Engineering of neocartilage in vitro is therefore a major target for autologous cartilage repair in arthritis. Previous analysis of neocartilage has targeted cellular organization and specific molecular components. However, the complexity of extracellular matrix (ECM) development in neocartilage has not been investigated by proteomics. To redress this, we developed a mouse neocartilage culture system that produces a cartilaginous ECM. Differential analysis of the tissue proteome of 3-week neocartilage and 3-day postnatal mouse cartilage using solubility-based protein fractionation targeted components involved in neocartilage development, including ECM maturation. Initially, SDS-PAGE analysis of sequential extracts revealed the transition in protein solubility from a high proportion of readily soluble (NaCl-extracted) proteins in juvenile cartilage to a high proportion of poorly soluble (guanidine hydrochloride-extracted) proteins in neocartilage. Label-free quantitative mass spectrometry (LTQ-Orbitrap) and statistical analysis were then used to filter three significant protein groups: proteins enriched according to extraction condition, proteins differentially abundant between juvenile cartilage and neocartilage, and proteins with differential solubility properties between the two tissue types. Classification of proteins differentially abundant between NaCl and guanidine hydrochloride extracts (n ‫؍‬ 403) using bioinformatics revealed effective partitioning of readily soluble components from subunits of larger protein complexes. Proteins significantly enriched in neocartilage (n ‫؍‬ 78) included proteins previously not reported or with unknown function in cartilage (integrin-binding protein DEL1; coiled-coil domain-containing protein 80; emilin-1 and pigment epithelium derived factor). Proteins with differential extractability between juvenile cartilage and neocartilage included ECM components (nidogen-2, perlecan, collagen VI, matrilin-3, tenascin and thrombospondin-1), and the relationship between protein extractability and ECM ultrastructural organization was supported by electron microscopy. Additionally, one guanidine extract-specific neocartilage protein, protease nexin-1, was confirmed by immunohistochemistry as a novel component of developing articular cartilage in vivo. The extraction profile and matrix-associated immunostaining implicates protease nexin-1 in cartilage development in vitro and in vivo. Molecular & Cellular Proteomics 9:1296 -1313, 2010.The cartilage of the mammalian skeletal system has two distinct roles. The epiphyseal cartilage of the growth plate drives endochondral bone growth, and the hyaline cartilage at the weight-bearing surfaces of bones facilitates joint articulation. In both environments, chondrocyte-regulated production, assembly, and turnover of the extracellular matrix (ECM) 1 are essential for the tissue to withstand compressive forces and respond to mechanical loading. The m...

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Cartilage proteoglycan aggregates. Electronmicroscopic studies of native and fragmented molecules

Cited by 47 publications

References 23 publications

Association of the Aggrecan Keratan Sulfate-rich Region with Collagen in Bovine Articular Cartilage

Association of the Aggrecan Keratan Sulfate-rich Region with Collagen in Bovine Articular Cartilage

The structure of proteoglycan aggregate determined by atomic force microscopy

Comprehensive Profiling of Cartilage Extracellular Matrix Formation and Maturation Using Sequential Extraction and Label-free Quantitative Proteomics

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