Acetylgalactosaminidase

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α-N-Acetylgalactosidase (α-NAGAL, EC 3.2.1.49) is a lysosomal exoglycosidase that can cleave terminal α-N-acetylgalactosamine residues from glycopeptides and glycolipids. In humans, the insufficient α-NAGAL activity can cause lysosomal storage disorders, Schindler and Kanzaki disease. Lysosomal storage disorder was first identified in 1987, which caused the accumulation of substrates in the tissues, which ultimately led to the development of clinical symptoms. In Schindler’s disease, the loss of functional α-NAGAL enzyme activity can lead to the accumulation of glycolipids and glycopeptides, which ultimately leads to neurological diseases and other diseases. The phenotype of Schindler disease has been divided into three classes. The type I disease is a serious infantile neurodegenerative disease. In Type II disease (also known as Kanzaki disease), the adult onset of the disease can lead to mild cognitive impairment and characteristic skin lesions, angiokeratoma. Type III disease shows a spectrum of symptoms including seizures, cardiomyopathy, and autism. There is currently no treatment for these diseases.

The structure of human α-NAGAL reveals the molecular basis of Schindler's disease. The collection of small molecule binding sites surrounding the active site provides the possibility to design molecules that may utilize the ligand binding characteristics revealed in the structure of human α-NAGAL. These molecules can be used as inhibitors or chemical chaperones of human α-NAGAL.

Fig 1. A. The reaction catalyzed by α-NAGAL (Clark, N.E.; Garman, S.C. 2009)

The relationship between α-NAGAL and α-GAL

In the human genome, the NAGA gene is closely related to the α-galactosidase A (GLA) gene, and they evolved from the same ancestral precursor. The corresponding proteins, α-NAGAL and α-GAL, have 46% amino acid sequence identity, but the substrate specificity is different. In fact, α-NAGAL was originally named α-GALB because it is considered to be an isozyme of α-GAL. In addition to removing the terminal α-GalNAc saccharides, human α-NAGAL protein also has some reactivity to substrates with α-galactose saccharides at the end.  

α-NAGAL and α-GAL proteins also have the ability to convert major blood group antigens. The α-NAGAL protein can enzymatically convert blood group A antigen to type O blood antigen, and the α-GAL protein can convert blood group B antigen to type O blood antigen. Since type O blood is a universal donor blood type, α-NAGAL and α-GAL enzymes have been used to convert type A, B and AB blood into type O blood. If α-NAGAL or α-GAL protein is defective, A or B blood group antigens will be processed abnormally. Another interesting aspect of α-GAL and α-NAGAL relates to the overlapping specificity of α-NAGAL, which can recognize and hydrolyze substrates containing terminal α-GalNAc saccharides and terminal α-galactose groups (less efficiency). However, the lack of α-GAL activity in Fabry disease cannot be compensated by α-NAGAL.

Overall description of the structure

Human α-NAGAL is a homodimer, each monomer contains 394 residues (not including the 17-residue signal sequence) and divided into two domains. Domain 1 forms a (β/α)₈ barrel and domain 2 contains eight antiparallel β strands in two β sheets. Like other members in family 27 and clan D glycoside hydrolases, its active site is located at the C-terminus of the β chain in the first domain. The active site is formed by loops C-terminal to six consecutive β strands, strands β1-β6. The residues that form the active site are strictly conserved between human and chicken α-NAGAL, indicating that these residues have strong evolutionary pressure.

The human α-NAGAL protein is negatively charged with an isoelectric point of 4.85, and the surface of the structure shows an obvious negative charge. Since this molecule usually functions in the low pH of the lysosome, the total charge on the molecule is approximately neutral at the lysosomal pH. Enzymatic studies have shown that the enzyme is most effective at pH 4.6. This molecule is a highly glycosylated and disulfide-rich glycoprotein. The wild-type protein contains five N-linked glycosylation sites (N124, N177, N201, N359, N385), four two disulfide bonds (C38-C80, C42-C49, C127-C158, C187-C209) and a free cysteine (C343).

According to its 46% sequence identity, human α-NAGAL and human α-GAL superimpose very well. The structure-based sequence alignment showed that even in the second domain where the sequence identity dropped to 23%, the secondary structure of the two proteins was very conserved.

Fig 2. Crystal structure of human α-N-acetylgalactosaminidase (Clark, N.E.; Garman, S.C. 2009)