By: Richard D. Cummings
It is well known that human cells produce a wide variety of glycans in the processes of biosynthesis (anabolism), and that cells also produce many glycosidases (glycoside hydrolases or glycohydrolases, GH) in processes that degrade glycans (catabolism). Such glycoside hydrolases can release a monosaccharide from the non-reducing end of a glycan, and may be termed an ‘exo-glycoside hydrolase’ or exo-glycosidase, but some release portions of the glycan by breaking bonds within it, and these are termed ‘endo-glycoside hydrolases’ or endo-glycosidases. Defects in glycoside hydrolases, e.g., α-L-iduronidase, glucocerebrosidase, etc., are associated with lysosomal storage disorders (LSDs), of which over 60 are known (1, 2). With that in mind, one might imagine that the glycoside hydrolases used by glycoscientists to study human and animal glycans might have a human or animal origin. But curiously that is seldom the case. Most of the glycoside hydrolases (exo- and endo-glycosidases) used in the field of glycosciences are derived from bacteria and fungi (and a few from viruses, animals and plants) (3-5). Such glycoside hydrolases are enzymes that are classified by the rules of the Enzyme Commission as EC 3.2.1.-. The CAZY database (Carbohydrate-Active EnZYmes) list 194 different GH family members. https://www.cazy.org/Glycoside-Hydrolases.html. Some of the enzymes are from organisms that live in extreme environments (extremophiles) and could offer expanded technological advantages (6). The glycoside hydrolases can recognize the linkage and monosaccharide residue and may result in either retention (retaining GH) or inversion (inverting GH) of the anomeric configuration (7, 8). For those glycoside hydrolases that are exo-glycosidases and release a monosaccharide, the released monosaccharide is often inhibitory to the enzyme. Even though we can imagine that some human glycans, such as heparin and heparan sulfate, might require human enzymes to degrade them, most of the heparinases or heparin lyases are from microorganisms, e.g., Pedobacter heparinus and Bacteroides species, and fungi, including Aspergillus (9). In any case, it is interesting that very few human glycoside hydrolases are used in the field of glycoscience.
Most of the enzymes from bacteria are in recombinant forms, but some from other sources are natural. Some of the companies that sell key glycosidases include New England Biolabs (NEB), Promega, Agilent, Sigma Aldrich, Lectenz Bio, and others. As an example, NEB currently offers many dozens of highly purified enzymes capable of degrading many glycans in a linkage specific manner. Metagenomic approaches are being successfully used to identify specific and useful enzymes throughout nature, providing a great diversity of novel enzymes to test. See for example modern studies on β-N-acetylgalactosamine-targeting glycosidases (10). But there are also a lot of other glycohydrolases used in the field, and they include amylases, cellulases, lactose, sucrase (invertase), chitinases, xylanases, etc. Some glycoside hydrolases have a low pH optimum (in the range of pH 4-5), but others are active at pH 7.0 and active over a relatively wide pH range. Also, glycoengineering is useful in refining and specifying useful enzyme activities (11). A few of the enzymes used in glycosciences are derived from animals and plants, such as β-galactosidases (β-1,3,4-galactosidase) from bovine testes (12), α-1,2,3,6-mannosidase from plants (Jack beans Canavalia ensiformis) (13), and α-1-3,4,6-galactosidase from coffee beans (Coffea arabica) (14).
There are several interesting historical facts about glycoside hydrolases as used in characterizing glycomolecules. One is that sialidases (neuraminidases) were first discovered as being in microbes, as from Vibrio cholera and influenza virus; the latter was found to have a neuraminidase which was first identified as a ‘receptor destroying enzyme’ by Macfarlane Burnet and Alfred Gottschalk in Melbourne, Australia in the 1940s, as it destroyed sialylated glycans recognized as a receptor by the hemagglutinin of the virus (15). Another curious and serendipitous finding was the enzyme now termed PNGase F (N-glycanase). It was accidentally discovered by John Elder and Stephen Alexander in 1982 at the Scripps Clinic as an unexpected activity in the supernatant of culture media from Flavobacterium meningosepticum (today known as Elizabethkingia meningoseptica) (16). It arose from their studies where they detected a bacterial contamination in bottles containing commercial preparations of carboxypeptidase A, and found that the culture fluid from the bacterial species produced an enzyme that could release N-glycans from glycoproteins, including of course, carboxypeptidase A. Because it was from Flavobacterium they named the enzyme Endo F. Their initial study showed the enzyme could remove N-glycans from all glycoproteins tested. Prior to this breakthrough there had already been discoveries of two other enzymes in 1974, now termed Endo H (17) from Streptomyces griseus and Endo D (18) from Diplococcus pneumoniae. By the way in the original publication, the name Endo H refers to the fact the enzyme releases ‘high’ molecular weight glycans, which include high mannose- and hybrid-type glycans (17).
References
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