A comparative structural bioinformatics analysis of inherited mutations in β-D-Mannosidase across multiple species reveals a genotype-phenotype correlation

Abstract

Background: Lysosomal β-D-mannosidase is a glycosyl hydrolase that breaks down the glycosidic bonds at the non-reducing end of N-linked glycoproteins. Hence, it is a crucial enzyme in polysaccharide degradation pathway. Mutations in the MANBA gene that codes for lysosomal β-mannosidase, result in improper coding and malfunctioning of protein, leading to β-mannosidosis. Studying the location of mutations on the enzyme structure is a rational approach in order to understand the functional consequences of these mutations. Accordingly, the pathology and clinical manifestations of the disease could be correlated to the genotypic modifications.

Results: The wild-type and inherited mutations of β-mannosidase were studied across four different species, human, cow, goat and mouse employing a previously demonstrated comprehensive homology modeling and mutational mapping technique, which reveals a correlation between the variation of genotype and the severity of phenotype in β-mannosidosis. X-ray crystallographic structure of β-mannosidase from Bacteroides thetaiotaomicron was used as template for 3D structural modeling of the wild-type enzymes containing all the associated ligands. These wild-type models subsequently served as templates for building mutational structures. Truncations account for approximately 70% of the mutational cases. In general, the proximity of mutations to the active site determines the severity of phenotypic expressions. Mapping mutations to the MANBA gene sequence has identified five mutational hot-spots.

Conclusion: Although restrained by a limited dataset, our comprehensive study suggests a genotype-phenotype correlation in β-mannosidosis. A predictive approach for detecting likely β-mannosidosis is also demonstrated where we have extrapolated observed mutations from one species to homologous positions in other organisms based on the proximity of the mutations to the enzyme active site and their co-location from different organisms. Apart from aiding the detection of mutational hotspots in the gene, where novel mutations could be disease-implicated, this approach also provides a way to predict new disease mutations. Higher expression of the exoglycosidase chitobiase is said to play a vital role in determining disease phenotypes in human and mouse. A bigger dataset of inherited mutations as well as a parallel study of β-mannosidase and chitobiase activities in prospective patients would be interesting to better understand the underlying reasons for β-mannosidosis.

Figure 1

3D structural models for wild-type (WT) sequences of a. goat, b. bovine, c. human and d. mouse. The associated ligands are shown as red spheres. All four WT β-mannosidase models are shown in the ribbon representation.

Figure 2

Human β-mannosidase active site. The residues that comprise the active site are in stick representation and are labeled. The catalytic nucleophiles (E457 and E554) are coloured red. The binding site residues are in magenta.

Figure 3

Mapping truncations to the human β-mannosidase protein. The extent of truncated mutants caused by nonsense, insertion and deletion mutations are shown as red, orange and green arrows, representing severe, moderate and mild phenotypes, respectively. Truncations in goat (broken arrow) and cow are shown below protein chain (blue rectangle). The purple and yellow arrow-heads represent the locations of the binding site residues and the catalytic nucleophiles, respectively. The corresponding single letter amino acid codes and ther residue numbers are given below the lower arrow-heads.

Figure 4

Proximity of substitution mutations to β-mannosidase active site. Amino acids are colored in red, magenta and yellow corresponding to the catalytic nucleophiles, ligand binding site residues and residues involved in substitutions, respectively. The active site residues and the residues involved in substutions are shown as sticks, while the rest of the protein is in ribbon representation. The model shown is that of WT human β-mannosidase.

Figure 5

The effect of substitutions on human β-mannosidase structure. a. R182W, b. G392E, c. S505P and d. R641H. Sections where the substitutions occur on the WT and mutant human β-mannosidase structures are shown. The WT and mutated residues are shown in red and green, respectively. The residues affected by substitutions are coloured turquoise. All amino acids involved in the mutations and/or affected by the substitutions are in stick representation, while the segments of the protein backbone are shown in ribbon representations (light grey). The dotted lines signify the interactions between the amino acid residues.

Figure 6

Conservation of β-mannosidase mutational residues across the four WT sequences. Multiple sequence alignment performed using ClustalX [26], shows a high level of sequence similarity, with conserved (*), conservatively substituted (:) and semi-conservatively substituted (.) residues. Sequence segments known to be mutational hot-spots are shown with the mutational residues highlighted in red.

Figure 7

Mapping inherited mutations in β-mannosidosis to the MANBA gene sequence. Missense and nonsense mutations are shown above the gene sequence, with the specific amino acid change indicated, while insertions and deletions are shown below the gene sequence, numbered in terms of the bases affected. The dark blue areas on the MANBA gene sequence represent sequence-derived mutational hot-spots.