Proteoglycan Detection

Proteoglycan Detection Kits

	Product	Catalog #	Price (USD)
	Glycosaminoglycans Assay Kit	6022	330.00
	Hyaluronan Competition Assay Kit	6048	389.00
	Hyaluronan Sandwich Assay Kit	6049	389.00
	Mouse Aggrecan Detection Kit	6058	399.00

Anti-Proteoglycan Antibodies

	Product	Catalog #	Price (USD)
	Mouse Anti-Mouse Aggrecan G1 Domain IgG1 Monoclonal Antibody, Clone 3F4G10	7166	260.00

Reagents for Sample Preparation

	Product	Catalog #	Price (USD)
	Papain	60224	23.00
	Pepsin	6011	15.00

Sulfated Glycosaminoglycans

Glycosaminoglycans (GAGs) are negatively charged polysaccharides located in most connective tissues and extracellular matrix (ECM), as well as on the surfaces of many cell types. Consisting of repeating core disaccharide units, GAGs are categorized into four types: heparin/heparan sulfate, chondroitin/dermatan sulfate, keratin sulfate and non-sulfated hyaluronan. Sulfated GAGs in the ECM exist as proteoglycan which typically consists of multiple glycosaminoglycan chains attached to a core protein. As a highly organized ECM, articular cartilage is composed of type II collagen, hyaluronan, link protein, and chondroitin sulfate-rich proteoglycans, which provide the osmotic resistance necessary for cartilage to resist compressive loads. Chondrex, Inc. provides a sulfated GAGs assay kit using the cationic dye, 1,9 dimethylmethylene blue (DMB) which binds to highly charged sulfated GAGs, not including hyaluronan. This kit utilizes an improved DMB solution minimizing interference with negatively charged contaminants, such as DNA and RNA, and chondroitin sulfate as an appropriate standard for the analysis of ECM in cartilage.

Table 1 provides a useful matrix to determine the ideal solubilization protocol for the analyte of interest.  In most cases, two different protocols will need to be used. For example, protocol A extracts GAGs, but fails to completely extract collagen and DNA, resulting in underestimated values (1,2). Protocol B solubilizes GAGs (3) and DNA (4), but degrades collagen. Finally, protocol C (Chondrex, Inc.'s protocol) can be used to measure GAGs and collagen (5), but not DNA.

Hyaluronans

Hyaluronan (HA: also called Hyaluronic acid) is a non-sulfated, unbranched glycosaminoglycan (GAG) composed of repeating disaccharide units of D-glucuronic acid and N-acetyl-D-glucosamine (6). Nearly all vertebrate cells can produce HA, with its expression closely linked to processes such as tissue expansion and cell movement (7). HA is primarily found in the extracellular and pericellular matrices, though it can also be present intracellularly. Its key biological roles include maintaining the elastoviscosity of fluid connective tissues such as joint synovial fluid and the vitreous humor in the eye, regulating tissue hydration and water transport, and organizing proteoglycans in the extracellular matrix. Additionally, HA is involved in receptor-mediated processes like cell detachment, mitosis, migration, tumor progression and metastasis, and inflammation. It also plays a vital role in binding water and lubricating movable parts of the body, such as joints and muscles (8). HA receptors on effector cells interact with HA and are involved in HA-related cell behavior and uptake (9). 

Aggrecan

Aggrecan, also known as cartilage-specific proteoglycan core protein or chondroitin sulfate proteoglycan 1 is a large proteoglycan vital for cartilage structure and function [10]. The protein core of aggrecan is approximately 250 kDa and is predominantly expressed in cartilage, particularly in articular cartilage, although it is also present in other connective tissues. Aggrecan exhibits a bottlebrush structure, in which chondroitin sulfate and keratan sulfate chains are attached to an extended protein core. This structure enables the formation of highly hydrated, gel-like assemblies that are critical for the load-bearing capacity and resilience of cartilage (11). Aggrecan contains three interglobular domains, G1, G2, and G3, and interacts with hyaluronic acid and link proteins (12).  The synthesis and degradation of aggrecan are being investigated with respect to their roles in cartilage deterioration during joint injury, disease, and aging (13-16). 

References

1. C. D. Hoemann, J. Sun, V. Chrzanowski, M. D. Buschmann, A multivalent assay to detect glycosaminoglycan, protein, collagen, RNA, and DNA content in milligram samples of cartilage or hydrogel-based repair cartilage. Anal Biochem 300, 1-10 (2002).
2. S. W. Sajdera, V. C. Hascall, Proteinpolysaccharide complex from bovine nasal cartilage. A comparison of low and high shear extraction procedures. J Biol Chem 244, 77-87 (1969).
3. R. W. Farndale, D. J. Buttle, A. J. Barrett, Improved quantitation and discrimination of sulphated glycosaminoglycans by use of dimethylmethylene blue. Biochem Biophys Acta 883, 173-177 (1986).
4. Y. J. Kim, R. L. Sah, J. Y. Doong, A. J. Grodzinsky, Fluorometric assay of DNA in cartilage explants using Hoechst 33258. Anal Biochem 174, 168-176 (1988).
5. K. von der Mark, M. van Menxel, H. Wiedemann, Isolation and characterization of new collagens from chick cartilage. Eur J Biochem 124, 57-62 (1982).
6. I. Hargittai, M. Hargittai. Molecular structure of hyaluronan: an introduction. Struct Chem 19, 697-717 (2008). 
7. V. Hascal, J. Esko. “Hyaluronan” in Essentials of Glycobiology (Cold Spring Harbor Laboratory Press, ed. 3, 2017). 
8. J. Necas, L. Bartosikova, P. Brauner, J. Kolar. Hyaluronic acid (hyaluronan): a review. Vet Med (Praha) 53(8), 397-411 (2008). 
9. B. Toole. Hyaluronan and its binding proteins, the hyaladherins. Curr Opin Cell Biol 2(5), 839-44 (1990). 
10. Doege, K.J., et al., Complete coding sequence and deduced primary structure of the human cartilage large aggregating proteoglycan, aggrecan. Human-specific repeats, and additional alternatively spliced forms. Journal of Biological Chemistry, 1991. 266(2): p. 894-902.
11. Roughley, P.J. and J.S. Mort, The role of aggrecan in normal and osteoarthritic cartilage. Journal of experimental orthopaedics, 2014. 1(1): p. 8.
12. Kiani, C., et al., Structure and function of aggrecan. Cell Research, 2002. 12(1): p. 19-32.
13. S. Larsson, L. S. Lohmander, A. Struglics, Synovial fluid level of aggrecan ARGS fragments is a more sensitive marker of joint disease than glycosaminoglycan or aggrecan levels: a cross-sectional study. Arthritis Res. Ther. 11, R92 (2009).
14. M. A. Pratta, J. L. Su, M. A. Leesnitzer, A. Struglics, S. Larsson, L. S. Lohmander, S. Kumar, Development and characterization of a highly specific and sensitive sandwich ELISA for detection of aggrecanase-generated aggrecan fragments. Osteoarthritis Cartilage 14, 702–713 (2006).
15. E. U. Sumer, B. C. Sondergaard, J. C. Rousseau, P. D. Delmas, A. J. Fosang, M. A. Karsdal, C. Christiansen, P. Qvist, MMP and non-MMP-mediated release of aggrecan and its fragments from articular cartilage: a comparative study of three different aggrecan and glycosaminoglycan assays. Osteoarthritis Cartilage 15, 212–221 (2007). 
16. S. Larsson, L. S. Lohmander, A. Struglics, An ARGS-aggrecan assay for analysis in blood and synovial fluid. Osteoarthritis Cartilage 22, 242–249 (2014).