Lysosomal exoglycosidases gradually degrade oligosaccharide chains of glycoconjugates (glycoproteins, glycolipids, glycosaminoglycans) in cell lysosomes. Defect in the activity of suitable lysosomal exoglycosidase stops degradation of oligosaccharide chains on sugar molecules not released by deficient exoglycosidase, and nondegraded oligosaccharide chains are stored in enlarged lysosomes. Enlarged lysosomes damage remaining cell structures and disturb the function of involved tissues, causing storage diseases. An increase in the activity of exoglycosidases in tissues and body fluids is observed in the reconstruction of damaged tissues. Exoglycosidase activity is an inexpensive and sensitive marker in diagnostics and monitoring of many diseases.
- lysosomal exoglycosidases
- fucosidase (FUC)
- β-D-galactosidase (GAL)
- β-D-glucuronidase (GLU)
- N-acetyl-β-hexosaminidase (HEX)
- α- and β-mannosidases (MAN)
1. Introduction: lysosomes
Inside lysosomes, more than 50 hydrolytic enzymes (glycosidases, proteases, lipases, nucleases, phosphatases, sulfatases, etc.) that are able to degrade all types of cell macromolecules are located. Lysosomal enzymes are active at acidic (pH ~5.0) water environment. High intralysosomal [H+] (about 100x higher than in cytoplasm) is maintained by vacuolar H+, V-type ATPase, located in the lysosomal membrane, which uses the energy of ATP hydrolysis to pump protons into lysosomes [1–3]. Lysosomal unique highly acidic environment creates some sort of protection for cytoplasmic components against noncontrolled autodigestion, additionally reinforced by integral proteins of the lysosomal membrane that are highly glycosylated to protect both lysosomal membrane and cytosolic elements against autodigestion [2, 4, 5]. Furthermore, some of the membrane glycoproteins function as specific receptors for molecules destined to degradation in lysosomes .
Designated for autodigestion, extracellular high-molecular substances reach lysosomes by endocytosis, pinocytosis, and phagocytosis . Intracellular high-molecular substances are digested by autophagy ; autophagy eliminates waste or damaged parts of the living cells. There are many types of autophagy: macroautophagy, microautophagy, chaperone-dependent autophagy, and specific autophagy. Autophagy may also be classified according to the digested material, e.g., mitophagy (digestion of mitochondria) or nucleophagy (digestion of nuclear debris) [8–10]. The best described is macroautophagy, where the cellular region destined for digestion is surrounded by the phospholipid membrane creating autophagosome. Then autophagosome merges with lysosome, where acid hydrolases degrade autosome contents into simple organic compounds, ready for utilization by the cell . Additionally, autophagy provides the cells with energy . Autophagy may be induced by hypoxia-caused stress, hunger, radiation, inflammation, and so on . In the case of pathological autophagy, cells exposed to intracellular toxins suffer from defective metabolism and die. Some of the researchers suspect that deficient autophagy may initiate many diseases such as diabetes or Alzheimer's disease , or even cancerogenesis. On the other hand, excessive autophagy may facilitate the survival of neoplastic cells during harmful conditions (e.g. chemotherapy). Therefore, autophagy in neoplasia may have dual biological sense .
2. Lysosomal enzymes
In autophagy, lysosomal acid proteases and glycosidases play a main role. Proteases cleave peptide bonds in the middle (endopeptidases) or outside (exopeptidases) of polypeptide chains. Main group of lysosomal proteolytic enzymes constitute cathepsins [14, 15], having aspartate (cathepsin D and E), cysteine (cathepsins B, C, H, K, and L), or serine (cathepsins A and G) in the active site [16, 17]. Proteases (PROT) (Figure 1) facilitate the action of three groups of glycosidases that gradually degrade tissue glycoconjugates (glycoproteins, glycolipids, and glycosaminoglycans):
Aminohydrolases as well as endo- and exoglycosidases create a sequence of reactions where the product of the previous enzyme is the substrate for the subsequent enzyme (Figure 1), and oligosaccharide is digested from reducing and nonreducing ends. When neuraminidase (NEU) releases N-acetylneuraminic acid (NANA) from the nonreducing ends of oligosaccharide chains, PROT degrade protein cores of glycoproteins, releasing reducing ends of oligosaccharides with attached asparagines. Oligosaccharides deprived of NANA are substrates for appropriate exoglycosidases depending on oligosaccharide composition. Oligosaccharides with β-D- galactose on non-reducing ends are substrates for β- galactosidase and oligosaccharides with α-L-fucose near reducing ends are substrates for α-L-fucosidase (Figure 1). Then, oligosaccharide chains are degraded by aspartylglucosaminidase (Asp-GlcNAc) that hydrolyses N-glycosidic bond between N-acetylglucosamine of the reducing end of oligosaccharide and asparagine remained from polypeptide as well as endo-N-acetylglucosaminidase (E-GlcNAc) releasing N-acetyloglucosamine from the reducing end of oligosaccharide chains. N-acetylhexosaminidase (NAG, N-acetyl-β-hexosaminidase (HEX)) releases N-acetyloglucosamine and N-acetylgalactosamine from a nonreducing end of the remaining part of oligosaccharide chains. Oligosaccharides containing mannose are substrates of α- and β – mannosidases (Figure 1). Lack or deficiency of a particular exoglycosidase blocks catabolism of oligosaccharide chains on a nondetached sugar residue . Disorders in the activity of lysosomal enzymes are closely related to autophagy and reflect intensity of development and course of many diseases, for example, infections, inflammations, cancers, heart diseases, Crohn’s disease, myopathy, liver diseases, and neurodegenerations. Autophagy is induced in cells by numerous factors: bacterial or viral infections, oxidative stress, and lack of nutrients. Some of the literature data also indicate the protective effect of autophagy [8, 20–23]. Increase in the activity of exoglycosidases in tissues [24–27] and body fluids [28–32] is observed in autophagy combined with the reconstruction of damaged tissues. In addition, determination of exoglycosidase activity is inexpensive and sensitive . In joint diseases (osteoarthritis, rheumatoid arthritis, and Lyme arthritis), progressive destruction of joint cartilages occurs. Destruction of cartilage is a multifactorial process caused by concerted action of lysosomal hydrolases (Figure 2). Proteases digest polypeptide chains of glycoconjugates exposing glycopeptides. Endoglycosidases (hyaluronidases, chondroitinases, keratanases, etc.) break down glycosidic bonds inside glycoconjugates and release oligosaccharide chains from the protein core. Lysosomal exoglycosidases, HEX, GAL, β-D-glucuronidase (GLU), and so on, release monosaccharides from the nonreducing terminals of oligosaccharide chains of glycoproteins, glycolipids, and glycosaminoglycans of synovial tissue, articular cartilage, and synovial fluid (Figure 2) .
2.1. Lysosomal exoglycosidases
Lysosomal exoglycosidases include GAL, GLU, FUC, HEX, as well as MAN. Among lysosomal exoglycosidases, the most active is
Human FUC is a glycoprotein occurring in different molecular forms. During separation on Sephadex G-200 column, FUC is eluted in two peaks: α-L-fucosidase I and α-L-fucosidase II. Both isoforms of α-L-fucosidase differ in molecular mass, pH optimum, and susceptibility on heat denaturation (both isoforms are thermolabile, but α-L-fucosidase I undergoes thermal inactivation in basic environment) .
Human GAL possesses three isoenzymes: A, B, and C (GAL C activity is small). Isoenzymes A and B absorb at 95% on concanavaline A (ConA) column, and at 60% on wheat germ agglutinin (WGA) column. Absorbance on ConA indicates the presence of mannose, and absorbance on WGA indicates the presence of N-acetylgalactosamine and N-acetylglucosamine in oligosaccharide chains of GAL .
Human MAN has three isoenzymes: A, B, and B2 that differ in sialic acid content and spatial arrangement of atoms in macromolecules . Human
2.1.1. Decrease in the activities of lysosomal exoglycosidases
Both deficiency and excessive HEX activity may have clinical significance. Inherited deficiency in
Absence or deficiency of
Decreased activity of α-L-fucosidase in breast tissue may be a predisposing factor for the appearance of breast cancer, because high levels of cell surface-associated α-L-fucose are related to neoplastic progression .
2.1.2. Increase in lysosomal exoglycosidase activities
Intensive inflammatory processes, for example tonsillitis, usually are accompanied by increase in lysosomal glycoconjugate catabolism . Hashimoto et al.  reported that pancreatic inflammation increases autophagy in the pancreatic inflamed cells. During autophagy, there is observed increase in the activity of lysosomal enzymes characteristic to the involved tissue. The most active of lysosomal exoglycosidases is
Determination of HEX in neoplastic tissues presents ambiguous results that depend on circumstances . Generally, in cancerous tissue, increase in the activity of hydrolytic enzymes including HEX should be observed. In tissues of benign neoplasm of human salivary gland a significant increase in HEX and its isoenzymes was observed, in comparison to healthy salivary gland . A significant increase in the activity of lysosomal enzymes (including HEX, HEX A, and HEX B) was reported in malignant brain tissue in comparison to brain tissues without neoplastic changes . But also significant decrease in HEX, HEX A, and HEX B activities in renal cancer tissue in comparison to healthy renal tissue was reported, followed by a significant increase in HEX and its isoenzymes in urine of neoplastic patients in comparison to healthy persons . Therefore, determination of urinary HEX and its isoenzymes may be particularly useful in diagnostics of neoplasms derived from renal epithelial cells of proximal contorted canalicules. Activity of urinary HEX and other exoglycosidases may be helpful in the diagnostics of pancreatic  and colon  cancers. Detection of HEX and its isoenzyme activity in stools may be used in elaboration of screening markers for detection of the colon cancer . Determination of HEX activity in serum and saliva may be used for the diagnostics and control of salivary gland tumors . The activity of lysosomal α-L-fucosidase (FUC)  reflects the intensity of degradation the α-L-fucose containing glycoproteins and glycolipids . The activity of β- galactosidase (GAL) reflects intensity of degradation glycoproteins, glycolipids and glycosaminoglycans containing galactose  and activity of β-glucuronidase (GLU) reflects intensity of glycosaminoglycans catabolism [61, 62].
Determination of the activities of FUC, GAL, and GLU may be applied for the diagnostics and monitoring of diseases proceeding with an increase in catabolism of oligosaccharide chains containing sugars released by appropriate exoglycosidases . Increase in the activity of
Activities of the lysosomal exoglycosidases in body fluids are good markers of neoplasms, inflammations, and infections. Determination of exoglycosidase activities in tissues may be helpful in establishing pathogenesis and treatment of some diseases, for example, nasal polyps. Nasal polyps are grape-shaped smooth structures, arising from the inflammatory nasal mucous membrane. Nasal polyps bulge to interior of the nose, restricting nasal patency . There are different pathogenesis theories of nasal polyps, however, none was satisfactorily confirmed, and the lack of understanding nasal polyp pathogenesis impedes therapy. It is known that untreated nasal polyps may cause intra- and extracranial complications. Currently used pharmacological and surgical treatments of nasal polyps do not provide satisfactory results . In nasal polyp tissue, a significant decrease in the concentration of activities of particular exoglycosidases was found in comparison to control, with simultaneous increase in specific activity of HEX A [73, 74]. A decrease in concentration of lysosomal exoglycosidases in nasal polyp tissue, without significant changes in their specific activities, denies the theory of full symptomatic inflammation in nasal polyp pathogenesis and may indicate neoplastic theory.
The activities of lysosomal exoglycosidases may be helpful in the selection of a proper method for treatment of hypertrophied and inflammatory palatal tonsils. Healthy palatal tonsils are important elements of immunological barrier against infections of the respiratory tracts [75, 76]. In the case of hypertrophy of lymphoidal tissue or chronic inflammation of palatal tonsils, otorhinolaryngologists very often face situations where palatal tonsils fail to serve as an immunological barrier and cause complications such as: impeded breathing and swallowing as well as speech disturbance. Palatal tonsils hypertrophy and inflammation are indication for tonsillotomy (trimming) or tonsillectomy (removal of palatal tonsils) [77, 78]. However, some otorhinolaryngologists claim that indications for tonsillo- and tonsillectomy should be limited, especially in younger children (6–7 years old), because the role of palatine tonsils and the possibility of surgical complications are not fully known [77–79]. Popko et al.  reported that the activity of lysosomal exoglycosidases in palate tonsils is independent of patients' age and she concluded that probably chronic inflammatory processes of the connective tissue of palate tonsils have the same intensity in childhood and in mature persons, and therefore she recommend tonsillectomy even in childhood.
3. Preparation of tissues and body fluids for determination of lysosomal exoglycosidases
Tissues immediately after resection, rinsing, and drying were frozen and stored at −80°C for a very long time. In homogenates and supernatant fluids, exoglycosidases should be determined without delay. Synovial fluids, urine, saliva, plasma, and serum may be stored at −80°C.
The above literature review indicates the activities of lysosomal exoglycosidases in tissues and body fluids as the markers for detection and monitoring of many human diseases.