A multi-species comparative structural bioinformatics analysis of inherited mutations in alpha-D-mannosidase reveals strong genotype-phenotype correlation

Abstract

Background: Lysosomal alpha-mannosidase is an enzyme that acts to degrade N-linked oligosaccharides and hence plays an important role in mannose metabolism in humans and other mammalian species, especially livestock. Mutations in the gene (MAN2B1) encoding lysosomal alpha-D-mannosidase cause improper coding, resulting in dysfunctional or non-functional protein, causing the disease alpha-mannosidosis. Mapping disease mutations to the structure of the protein can help in understanding the functional consequences of these mutations and thus indirectly, the finer aspects of the pathology and clinical manifestations of the disease, including phenotypic severity as a function of the genotype.

Results: A comprehensive homology modeling study of all the wild-type and inherited mutations of lysosomal alpha-mannosidase in four different species, human, cow, cat and guinea pig, reveals a significant correlation between the severity of the genotype and the phenotype in alpha-mannosidosis. We used the X-ray crystallographic structure of bovine lysosomal alpha-mannosidase as template, containing only two disulphide bonds and some ligands, to build structural models of wild-type structures with four disulfide linkages and all bound ligands. These wild-type models were then used as templates for disease mutations. All the truncations and substitutions involving the residues in and around the active site and those that destabilize the fold led to severe genotypes resulting in lethal phenotypes, whereas the mutations lying away from the active site were milder in both their genotypic and phenotypic expression.

Conclusion: Based on the co-location of mutations from different organisms and their proximity to the enzyme active site, we have extrapolated observed mutations from one species to homologous positions in other organisms, as a predictive approach for detecting likely alpha-mannosidosis. Besides predicting new disease mutations, this approach also provides a way for detecting mutation hotspots in the gene, where novel mutations could be implicated in disease. The current study has identified five mutational hot-spot regions along the MAN2B1 gene. Structural mapping can thus provide a rational approach for predicting the phenotype of a disease, based on observed genotypic variations.

Figure 1

Flowchart of the three-step modeling procedure used in this study. The three main steps involved in building a structural model are described, with reference to query sequence, template sequences and template structure.

Figure 2

Structural model of wild-type bovine α-mannosidase. The structure is shown in Cα trace representation (in grey) with chains B and E highlighted in magenta and gold respectively. The bound ligands present in the template structure are shown as green spheres. Disulfide bonds in the template structure are in yellow ball-and-stick representation, with new disulfide bonds in the model in red.

Figure 3

WEBLOGO alignment of putative MAN2B1 pro-peptide sequences from six mammalian species. The alignment represents sequences from human, bovine, cat and guinea pig, along with two other mammalian species, mouse and macaque.

Figure 4

Structural model of WT bovine α-mannosidase showing the location of all mutations studied in different species. The spheres denote positions of truncation mutations. Amino acids involved in substitution mutations are shown in stick representation. The catalytic zinc ion is shown as a blue sphere. Mutations are coloured red, green and orange, representing harmful, viable and mild phenotypes, respectively.

Figure 5

Part of the enzyme active site, with a high concentration of mutations leading to lethal phenotypes. Several aromatic residues surround the active site and potentially are involved in binding of the Tris ligand. Tris (white) is held in position by the catalytic nucleophile, D196 (blue) via a zinc ion (orange). R220 (green) is directly involved in ligand binding, along with H72 (purple) and Y380 (magenta). The aromatic side chains of F320 (red) and Y84 (yellow) interact to hold the structure together. These interactions are shown in dotted lines.

Figure 6

Mapping truncation mutations to the different chains of the human α-mannosidase protein. The extent of nonsense, insertion and deletion mutants are shown as red, orange and green arrows, represent lethal, moderate and mild phenotypes, respectively.

Figure 7

Conservation analysis of mutations across the four species. Alignment of the wild-type sequences from all four species 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. Sequence names and numbers are from Swiss-Prot.

Figure 8

Active site of bovine lysosomal α-mannosidase. The Tris ligand (labeled Trs) is shown in orange, the Zn²⁺ ion in purple and all the active site residues in red (ball-and-stick representation). These active site residues represent a structural hot-spot region, for the lethal phenotype.

Figure 9

Location of all non-splicing mutations on the MAN2B1 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 grey areas represent sequence-derived mutational hot-spot regions.