A multi-species comparative structural bioinformatics analysis of inherited mutations in a-D-Mannosidase reveals strong genotype-phenotype correlationJaved Mohammed Khan and Shoba RanganathanMacquarie University, Sydney, Australiajkhan@cbms.mq.edu.au
a-D-MannosidaseLysosomal a-mannosidase acts to degrade N-linked oligosaccharides or mannose-rich compounds.The deficiency of this enzyme results in a recessively inherited lysosomal storage disease, called a-mannosidosis.Mutations in the gene (MAN2B1) encoding lysosomal a-D-mannosidase cause the disease.Structure of bovinea-D-mannosidase shows five chains held togetherby four disulfide bonds.Missense, nonsense, insertions, deletions and also some splicing mutations.
Phenotypes and species affectedCoarse face, prominent forehead, mental retardation, skeletal malformation, hearing loss and cerebral dysfunction leading to paralysis and death1. A severe infantile formLeads to early deathA mild juvenile form Survival into adult lifeDomestic cats, cattle and guinea pigs.First characterized in humans by Ockerman in 19672.Malm D, Nilssen : Alpha-mannosidosis. Orphanet J Rare Dis. 2008, 3:21. Ockerman PA. A generalised storage disorder resembling Hurler's syndrome. Lancet. 1967;2:239. Artemisia annua
Why study a-D-mannosidosis?It can be comparatively well studied due to its occurrence across various species.Clinicians, geneticists and molecular biologists have not been able to correlate the genotypic mutations with the observed phenotype1, 3. The high phenotypic variability, has so far prevented adoption of a standardized clinical typing.a-mannosidosis occurs in 1 of 500,000 live births1.Errors of lysosomal metabolism occur in approximately 1:5000 live births.This approach can be extended to other lysosomal disorders.Malm D, Nilssen : Alpha-mannosidosis. Orphanet J Rare Dis. 2008, 3:21.Lyons MJ, Wood T, Espinoza L, Stensland HM, Holden KR: Dev Med Child Neurol 2007, 49:854-857.
Our approachUse protein structure to unravel how the mutations affect the function of the enzyme, especially with respect to the enzyme active site, to explain observed phenotypes.Data sources for mutationsOMIM: Online Mendelian Inheritance in ManPublications from PubMed. OMIA: Online Mendelian Inheritance in AnimalsSequence informationGenBank, Swiss-ProtSequence alignment and visualizationCLUSTAL, WEBLOGO3D structural modelingMODELLER3D structure assessmentBiotech Validation Suite
Issues related to modelling The X-ray crystal structure for bovine lysosomal a-D-mannosidase (PDB ID: 1O7D)4 lacks: Two of the four vital disulfide bonds (as annotated by Swiss-Prot). A few structurally and functionally important residues.
MAN2B1 produces a five chain protein.
Existence of 11 essential ligands.
Presence of cleavable signal peptide (50 AA) in database sequences and water molecules in 3D structure.
Swiss-Prot and WEBLOGO depict a well conserved propeptide region. Heikinheimo P, et al.: J Mol Biol 2003, 327:631-644.
What is the solution?A single comprehensive molecular modeling procedure.
Addresses all the issues related to a-D-mannosidase modeling. Rebuilt the two missing disulfide bridges along with all the existing ligands.
Assessment of the Wild-Type structural models High degree of similarity between target and template sequences and strict modeling protocols adopted.
Excellent RMSD and WHATIF Z-Scores.
Above average PROVE Z-Score and PROCHECK validation results.
Generation of high quality WT models.
Mapping mutations to WT structural models We have constructed a mutation map mapping available mutations in the context of the enzyme active site to understand where the observed mutations occur Most mutations with lethal phenotypes are located in and around the active site. Thereby affecting the functionality of the enzyme.
Genotype-Phenotype correlation The three phenotypes correspond to Type 3, Type 2 and Type 1 clinical phenotypes described by Malm and Nilssen1. Mapped all truncation mutations to different chains of human WT protein. Malm D, Nilssen : Alpha-mannosidosis. Orphanet J Rare Dis. 2008, 3:21.
Prediction of potentially harmful mutations Highly conserved sequence across all species. Almost all mutated residues causing fatal/harmful phenotypes are highly conserved. All these positions can be considered potential disease-causing mutations for all mammals represented here. Mutations in residues comprising the active site of the enzyme could have serious effectsThis residue set represents a structurally-derived mutation hot-spot.
Sequence-based mutational hot-spot regions in the MAN2B1 gene Mutations are scattered along the length of the gene. Mutations seem to cluster into groups over segments of varying sequence length called mutational hot-spot regions. Five distinct mutational hot-spot regions with lengths varying from 117 to 606 nucleotides. Residues coded for by the nucleotides within the range 961-1204 are most likely to undergo mutations. Occurrence of a harmful mutation is most likely to be between 157-323, 562-679 and 961-1204 hot-spot regions due to their close proximity to the active site.
Significance of this studyCan be used as a predictive approach for detecting likely -mannosidosis across various species.
Novel prediction protocol for new disease mutations related to -mannosidosis.
Approach can be extended to other inherited disorders.
Provides a way for detecting mutation hotspots in the gene, where novel mutations could be implicated in disease.
A rational approach for predicting the phenotype of a disease, based on observed genotypic variations.
ConclusionsEstablishes a significant correlation between the genotype and the phenotype of the disease.
Highlights the effect of disease mutations on protein structure and forms the basis for understanding the molecular determinants for phenotypic variations.
High degree of mutational heterogeneity of -mannosidosis is comparable to that observed in many other lysosomal disorders.
Suggests that rather than drug/inhibitor design, this disease could be tackled through gene therapy.
This study could play a vital role in developing therapies for inherited diseases.
AcknowledgementsProf. Shoba Ranganathan.Macquarie University for MQRES scholarship.Colleagues and friends at Macquarie University.The InCoB 2009 program and organising committees.