**3. The characterization and function of lysosomal glycosidases**

The main exoglycosidases in tissues, serum and synovial fluid of humans are:

N-acetyl-β-hexosaminidase (HEX), β-glucuronidase (GluA), β-galactosidase (GAL), αmannosidase (α-MAN), and α-fucosidase (FUC). N-acetyl-β-hexosaminidase (EC 3.2.1.52, HEX, NAG) is the most active enzyme of the lysosomal exoglycosidases (Popko et al. 2006). HEX has several isoenzymes: A, B, S, C, I1, I2. HEX A and S are thermolabile and B, P, I1, I2, thermostable. In humans there are two major isoenzymes of hexosaminidase: HEX A (αβ), and HEX B (ββ). Both isoenzymes recognize terminal N-acetylglucosamine and Nacetylgalactosamine, but only HEX A recognizes 6-sulfated residues of these sugars. HEX A represents (an average) 48% of total HEX activity in serum, and 52% of total HEX activity in synovial fluid (Popko et. al.2006).

The hexosaminidase S (HEX S) is of minor importance, as it constitutes less than 0.02% of HEX activity (Ikonne et al. 1975)), and can be detected in patients with Sandhoff disease (Yamanak et al. 2001). The function of the HEX S is not well understood, but it is probably involved in the degradation of GAGs.

The protein moiety of lysosomal exoglycosidases is synthesized in the rough endoplasmic reticulum, and transported to lysosomes thought the endoplasmatic reticulum and Golgi apparatus (Zwierz et al.1999). Some of the lysosomal enzymes are secreted from the cell into the extracellular fluid. Another route for the secretion of lysosomal enzymes is from the lysosomes via the endosomes and Golgi compartment to the cell surface and extracellular fluid. The release of exoglycosidases is regulated by a small Ras-related GTP-binding protein Rho p21 (Rho proteins control the polymerization of actin into filaments and govern the organization of body filaments into specific types of structures). The release of exoglycosidases from mast cells has shown to be induced by an IgE mediated increase in intracellular Ca²+ (Zwierz et al. 1999).

Exoglycosidases activity of knee synovial fluid and serum of healthy humans is presented in Fig. 2.

The exoglycosidases activity, is higher in synovial fluid than in serum. Levels of the HEX activity are constant in serum of healthy humans up to 40 years of age, whereas in older people (more than 40 years of age) the level of HEX activity significantly increases.

The substrates for exoglycosidases in articular cartilage include cell surface and extracellular matrix glycoproteins as well as glycosaminoglycans: chondroitin 4-sulfate, chondroitin 6 sulfate, hyaluronic acid, keratin sulfate, and dermatan sulfate (Winchester 1996; Stypułkowska et al. 2004).

Lysosomal Glycosidases in Degradation of Human Articular Cartilage 101

Pugh and Walker (Pugh 1961), using histochemical techniques, reported that the source of HEX activity in synovial fluid is from cells of the synovial membrane. Others (Shikhman et al. 2000; Ortutay et. al. 2003) have suggested that chondrocytes of RA patients activated by IL 1β (interleukin-1β) may be a source of HEX activity in synovial fluid. Relating to this, it has been observed that damage to the joint reduces the volume of cartilage and increases a

Profiles of the exoglycosidases in the synovial membrane of the knee joint of patients with

Fig. 4. The activity of exoglycosidases in the synovial tissue of the knee joint of patients with

Normal and inflamed synovial tissues have similar patterns of exoglycosidases activity with a significant predominance of HEX activity (Popko et.al.2006). HEX activity in the synovial tissue of RA and JIA patients was approximately 10-fold higher than in the synovial tissue of the reference group. The increase in activity of GluA, GAL, MAN and FUC in synovial tissue of RA and JIA patients (in comparison with reference groups) was moderate, i.e. no

Synovial fibroblast-like cells and chondrocytes may be regarded as a source of mediators of joint destruction in RA and JIA. Synoviocytes and chondrocytes secrete proteolytic enzymes and exoglycosidases, especially HEX, that are crucial for the degradation of cartilage. The destructive phenotypes of the synovial fibroblasts-like cell and chondrocytes in RA are probably regulated by inflammatory cytokines released by the pannus connected to the

**4. The localization of exoglycosidases in joint tissues** 

proliferation of synovial membrane (Fig. 3).

RA and JIA.

cartilage.

more than doubled.

RA, JIA and a control group are presented in Fig. 4.

Fig. 2. Activity of exoglycosidases (nmol/ml/min) in synovial fluid and serum of healthy humans.

Exoglycosidases degrade glucoconjugates within the lysosome at an optimum pH ranging from 4.3 to 5.5 (Zwierz et. al. 1989; Marciniak et. al. 2006). Investigation of the pH dependence showed that HEX is active at pH 4.2 to 5.6 with optimum at pH 4.7, and βglucuronidase is active between pH 3.4 and 5.6, with optimum activity at pH 4.5 (Marciniak et al. 2006).

Fig. 3. Typical knee of a patient with RA. Showing destruction areas of articular cartilage and hypertrophy of synovial membrane.

Fig. 2. Activity of exoglycosidases (nmol/ml/min) in synovial fluid and serum of healthy

Fig. 3. Typical knee of a patient with RA. Showing destruction areas of articular cartilage

and hypertrophy of synovial membrane.

Exoglycosidases degrade glucoconjugates within the lysosome at an optimum pH ranging from 4.3 to 5.5 (Zwierz et. al. 1989; Marciniak et. al. 2006). Investigation of the pH dependence showed that HEX is active at pH 4.2 to 5.6 with optimum at pH 4.7, and βglucuronidase is active between pH 3.4 and 5.6, with optimum activity at pH 4.5 (Marciniak

humans.

et al. 2006).
