The human lysosomal enzyme α-NAGAL (E.C. 220.127.116.11) removes terminal α-GalNAc monosaccharides from glycolipids and glycoproteins (primarily O-linked sugars attached to serine and threonine residues) (). Deficiency in α-NAGAL leads to the lysosomal storage disorder Schindler disease, first identified in 1987.1,2,3
In lysosomal storage disorders, loss of enzyme activity in a patient leads to the accumulation of substrate in the tissues, which ultimately leads to the development of clinical symptoms. In Schindler disease, loss of functional α-NAGAL enzyme activity causes accumulation of glycolipids and glycopeptides, which ultimately results in neurologic and other pathologies.3
Schindler disease phenotypes have been grouped into three classes. The Type I disease is a severe infantile neurodegenerative disorder.4,5
In Type II disease (also known as Kanzaki disease), adult onset of the disease leads to mild cognitive impairments and a characteristic skin lesion, angiokeratoma.6-9
Type III disease displays a spectrum of symptoms including seizures, autistic disorders, and/or cardiomyopathy.8,10,11
There is no treatment for these disorders.
α-NAGAL reaction and overall structure
In the human genome, the NAGA
gene is most closely related to the α-galactosidase A (GLA
) gene, having evolved from the same ancestral precursor.12
The corresponding proteins α-NAGAL and α-GAL have 46% amino acid sequence identity, but different substrate specificities. The human α-NAGAL protein, in addition to removing terminal α-GalNAc saccharides, has some reactivity toward substrates with terminal α-galactose saccharides. In fact, the enzyme was originally named α-GAL B and was thought to be an isozyme of α-GAL.13
In contrast, the human α-GAL protein (E.C. 18.104.22.168) removes terminal α-galactose saccharides from substrates, but it shows no enzymatic activity toward substrates with terminal α-GalNAc saccharides. Deficient α-GAL enzyme activity leads to the accumulation of glycoconjugate substrates, primarily globotriaosylceramide (Gb3
), which results in Fabry disease.14
Because the GLA
gene coding for the human α-GAL protein resides on the X-chromosome, Fabry disease is inherited as an X-linked disorder.
The α-NAGAL and α-GAL proteins also have the ability to convert major blood group antigens.15
The α-NAGAL protein can enzymatically convert the blood group A antigen into blood group O antigen, and the α-GAL protein is able to convert the blood group B antigen into the blood group O antigen. Because type O blood is the universal donor blood type, the α-NAGAL and α-GAL enzymes have been used to seroconvert types A, B, and AB blood into type O blood.16
Individuals with defects in their α-NAGAL or α-GAL proteins abnormally process blood group A or B antigens.17,18
In Fabry disease, human α-GAL deficiency results in the accumulation of its substrates with terminal α-galactose residues, and to a lesser extent, their precursors.14
However, in the Schindler diseases, the substrates that accumulate do not contain terminal α-GalNAc saccharides, but instead contain sialic acid- and galactose-terminal saccharides, similar to those in the lysosomal storage disorders sialidosis and galactosialidosis.19,20
It has been suggested that the α-NAGAL glycoprotein is part of a larger macromolecular assembly (with α-neuraminidase, β-galactosidase, and protective protein), and that loss of functional α-NAGAL leads to malfunction of the complex in the lysosome.3
A second possibility is that in the absence of functional α-NAGAL in the lysosome, other glycosidases such as α-neuraminidase might work in a reverse reaction, acting as glycosyltransferases in the presence of large amounts of enzymatic product.21
Another interesting aspect of α-GAL and α-NAGAL relates to the overlapping specificity of α-NAGAL, which can recognize and hydrolyze substrates with terminal α-GalNAc saccharides and (less efficiently) those with a terminal α-galactose moiety. However, the absence of α-GAL activity in Fabry disease is not compensated by α-NAGAL.
Previously, we reported the structures of the human α-GAL22
and chicken α-NAGAL enzymes.23
Those structures allowed us and others to make homology models of the human α-NAGAL enzyme in an effort to understand the molecular defects resulting in disease.23-25
However none of the homology models clarified the above issues. To address these and to establish the molecular basis for Schindler diseases, we performed structural studies of the human α-NAGAL enzyme. Using a recombinant insect cells, we expressed the functional wild type α-NAGAL glycoprotein as well as mutants lacking each of the five N-linked glycosylation sites. We measured the enzymatic activities of our wild type and mutant enzymes and determined the structure of human α-NAGAL to 1.9 Å resolution, revealing the mechanism of the enzyme. To determine the binding specificity and catalytic mechanism of human α-NAGAL, we determined crystallographic complexes with two catalytic products (the α-galactose and α-GalNAc monosaccharides) and a covalent intermediate bound in the enzyme’s active site. To better understand how individual mutations in the NAGA gene lead to Schindler or Kanzaki disease, we analyzed the respective defective enzymes in light of the three-dimensional structure. Overall, these results will lead to better understanding of the molecular defects in Schindler disease and will provide insight into lysosomal storage diseases and protein folding diseases.