Javed Mohammed Khan and Shoba Ranganathan

1. A multi-species comparative structural bioinformatics analysis of inherited mutations in a-D-Mannosidase reveals strong genotype-phenotype correlation Javed Mohammed Khanand Shoba Ranganathan Macquarie University, Sydney, Australia jkhan@cbms.mq.edu.au

2. E C A D B a-D-Mannosidase • Lysosomal 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 bovine a-D-mannosidase shows five chains held together by four disulfide bonds. • Missense, nonsense, insertions, deletions and also some splicing mutations.

3. Phenotypes and species affected • Coarse face, prominent forehead, mental retardation, skeletal malformation, hearing loss and cerebral dysfunction leading to paralysis and death1. • A severe infantile form • Leads to early death • A mild juvenile form • Survival into adult life • Domestic cats, cattle and guinea pigs. • First characterized in humans by Ockerman in 19672. Artemisia annua • Malm D, Nilssen Ø: Alpha-mannosidosis. Orphanet J Rare Dis. 2008, 3:21. • Ockerman PA. A generalised storage disorder resembling Hurler's syndrome. Lancet. 1967;2:239.

4. 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 Ø: 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.

5. Our approach • Use 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 mutations • OMIM: Online Mendelian Inheritance in Man • Publications from PubMed. • OMIA: Online Mendelian Inheritance in Animals • Sequence information • GenBank, Swiss-Prot • Sequence alignment and visualization • CLUSTAL, WEBLOGO • 3D structural modeling • MODELLER • 3D structure assessment • Biotech Validation Suite

6. 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 cleavablesignal 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.

7. 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.

8. 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.

9. Mapping mutations to WT structural models • We haveconstructed 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.

10. 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 Ø: Alpha-mannosidosis. Orphanet J Rare Dis. 2008, 3:21.

11. 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 effects • This residue set represents a structurally-derived mutation hot-spot.

12. 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.

13. Significance of this study • Can be used as a predictive approach for detecting likely α-mannosidosis across various species. • Novel prediction protocol for new disease mutations related to α-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.

14. Conclusions • Establishes 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 α-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.

15. Acknowledgements • Prof. Shoba Ranganathan. • Macquarie University for MQRES scholarship. • Colleagues and friends at Macquarie University. • The InCoB 2009 program and organising committees.

16. Questions ?