Type A & B Niemann-Pick diseaseand acid sphingomyelinase structure2023/01/12
B4 Ryota Matsukawa

Contents
1. Sphingomyelin and Niemann-Pick disease (NPD) type A and B
2. Therapy for type A and B NPD
3. Crystal structures of acid sphingomyelinase
4. Summary

Contents
1. Sphingomyelin and Niemann-Pick disease (NPD) type A and B
2. Therapy for type A and B NPD
3. Crystal structures of acid sphingomyelinase
4. Summary

Sphingomyelin and acid sphingomyelinase (ASM)
Sphingomyelin• Phosphate diester of phosphocholine and ceramide• Ceramide: sphingosine + fatty acid
• A major component of the outer leaflet of cell membranes

Wikipedia sphingomyelin
https://en.wikipedia.org/wiki/Sphingomyelin

The Cell: The Histology Guide
https://www.histology.leeds.ac.uk/cell/plasma_membrane.php• Sphingomyelin is hydrolyzed by acid sphingomyelinase (ASM)

Zhou, YF. et al., Nat. Commun., 2016, 7, 13082.

Acid sphingomyelinase (ASM)
Pathway to hydrolysis of sphingomyelin

J. Kornhuber, et al., Biol. Chem., 2015, 396, 707.

Niemann pick disease (NPD) type A and B
Niemann-Pick disease type A and B• Acid sphingomyelinase deficiency (ASMD)･･･ Abnormal accumulation of sphingomyelin in lysosomes or cell membrane
in liver, spleen, lung and central nerve system due to mutations in SMPD1
which encoding ASM• Clinical features and pathology･･･ Type A: Hepatosplenomegaly (肝脾腫), hypotonia (筋緊張低下),
progressive neurological symptoms
Type B: Hepatosplenomegaly (肝脾腫), liver failure (肝不全),
decreased pulmonary function (肺機能の低下)
Splenomegaly (脾腫)Jesús, V. et al., Br. J. Haematol., 2016, 172, 840.

Foam cells (泡沫細胞)

M. Cerón-Rodríguez et al., Ann. Hepatol., 2019, 18, 613.
E.H. Schuchman, et al., Mol. Genet. Metab., 2017, 120, 27.

Contents
1. Sphingomyelin and Niemann-Pick disease (NPD) type A and B
2. Therapy for type A and B NPD• Bone marrow transplantation• Enzyme replacement therapy• Gene therapy
3. Crystal structures of acid sphingomyelinase
4. Summary

Bone marrow transplantation (BMT)
Bone marrow transplantation for
ASM knock-out mouse model

Dotted line: Controls (+/+)
Hatched line: ASMKO controls (-/-)
Solid line: Transplanted ASMKO• Sphingomyelin levels in the spleen and liver
decreased significantly.
• The effect on the brain was moderate and theprogressive neurological disease was not prevented.
S.R. Miranda, et al., Transplantation, 1998, 65, 884.

Enzyme replacement therapy (ERT)
Olipudase alfa (Xenpozyme) for ASMKO mouse
Olipudase alfa: Recombinant human ASM
Sphingomyelin content in tissues+/+: Normal control, -/-: ASMKO control, ERT: Treated ASMKO• Histological improvement was observed by infusion of rhASM.

SRP. Miranda, et al., FASEB J., 2000, 14, 1988.

Enzyme replacement therapy (ERT)
Olipudase alfa (Xenpozyme) for NPD type B patients
Olipudase alfa: Recombinant human ASM• Accumulated sphingomyelin was debulked and histologicalimprovement was observed by treatment of Olipudase alfa.
M. P. Wasserstein, et al., Mol. Genet. Metab., 2015, 116, 88.

Gene therapy
Hematopoietic stem cell gene therapy+/+: Normal control, -/-: ASMKO control, GT: Treated ASMKO• Tissues from the treated mice show markedly less lipid accumulation
than ASMKO control.
SRP. Miranda, et al., Gene Ther., 2000, 7, 1768.

Gene therapy
Hematopoietic stem cell gene therapy• It has been reported that Purkinje cells are reduced in thecerebellum in the ASMKO mouse model.
K. Horinouch, et al., Nat. Genet., 1995, 10, 288-293.
Gene therapy
Purkinje cell immunohistochemistrya. 5-month-old ASMKO b. Treated ASMKO c. Normal control• Purkinje cells are present throughout thecerebella of the treated mice.
SRP. Miranda, et al., Gene Ther., 2000, 7, 1768.

Contents
1. Sphingomyelin and Niemann-Pick disease (NPD) type A and B
2. Therapy for type A and B NPD
3. Crystal structures of acid sphingomyelinase
4. Summary

ASM structures
Diagram of ASM domains• Yellow: Saposin domain (N-terminal)
Sphingomyelin activator that recognize and locate lipids on
the active site
• Orange: Proline-rich linker
The connection between the saposin and the catalytic domain• Blue: Metallo-dependent phosphatase catalytic domain
Active site for hydrolysis of sphingomyelin• Green: C-terminal domain (ill-defined)

Zhou, YF. et al., Nat. Commun., 2016, 7, 13082.

ASM structures
Overall structure of ASM

Catalytic domain

N-Glycosylation

Zhou, YF. et al., Nat. Commun., 2016, 7, 13082.

ASM structure: Domain interactions
Interactions between the saposin domain
and catalytic domain
Saposin domain

Catalytic domain• H3 helix in saposin domain extensively contacts with β1-α1, β2-α2,β3-α3, β5-α5 loop.
• Many mutations occur on the interface residues, such as L137P in H3,P323A on the β3-α3 loop, ΔF390 on the β5-α5 loop.
Zhou, YF. et al., Nat. Commun., 2016, 7, 13082.

ASM structure: Domain interactions
Interactions between the proline linker
and catalytic domain

Proline-rich linker

Catalytic domain• The proline-rich linker is stabilized by hydrophobic interactions between
W174, I176, L178 in the proline-rich linker and α5, α6 helices in thecatalytic domain.
Zhou, YF. et al., Nat. Commun., 2016, 7, 13082.

ASM structure: Domain interactions
Interactions between the proline linker
and C-terminal domain

Catalytic domain

C-terminal domain• The C-terminal domain contributes to stabilization of the active site,
especially β7-β8 loop.
• It has been reported that ΔT592 mutation causes severe ASMD.
→ ΔT592 mutation has a severe impact on ASM folding?
Rodriguez-Pascau, L. et al., Hum. Mutat., 2009, 30, 1117.
Zhou, YF. et al., Nat. Commun., 2016, 7, 13082.

ASM structure: Zinc and phosphocholine binding in the active sitea. Active site of holo ASM structureb. Phosphocholine bound structure• Both zincs have trigonal bipyramidal geometry.
• A water molecule bridges between Zn1 and Zn2 and is stabilizedby hydrogen bonding to the main chain carbonyl of H457.
• Oxygen (O1) in the phosphoryl group replaces the position of
the water molecule in the holo ASM structure.
Zhou, YF. et al., Nat. Commun., 2016, 7, 13082.

Hydrolysis of sphingomyelin
Mechanism of hydrolysis of sphingomyelin• The coordinated water is deprotonated to hydroxide due to the activationby two zinc ions, and the hydroxide attacks on the phosphorus ofsphingomyelin.
• The phosphorus oxygen on the ceramide side extracts a proton from H319.
• Mutation of H319Y causes severe ASMD.
Sikora, J. et al., Ann. Hum. Genet., 2003, 67, 63.
Zhou, YF. et al., Nat. Commun., 2016, 7, 13082.

Docking model of sphingomyelin on ASM
a. Surface residues around C-16 sphingomyelinb. Electrostatic potential maps
Saposin domain

Catalyticdomain

Sphingomyelin• The hydrophobic ceramide chains in sphingomyelin is accommodated inthe saposin concave surface which is rich in hydrophobic residues andnot highly charged.

Zhou, YF. et al., Nat. Commun., 2016, 7, 13082.

Mutation in ASM
a. Distribution of mutations
on ASM sequenceb. Distribution of mutations
on 3D structure• 82% of mutations are located in the catalytic domain.
• Those mutations are categorized into two groups, according to theirdisruptive effects on catalytic activity and protein folding.

Zhou, YF. et al., Nat. Commun., 2016, 7, 13082.

Mutation affecting catalytic activity
D278A, H329Y, H425R mutations

Zhou, YF. et al., Nat. Commun., 2016, 7, 13082.

Mutation affecting protein folding
ΔT592 mutations

Catalytic domain

C-terminal domain

Zhou, YF. et al., Nat. Commun., 2016, 7, 13082.

Contents
1. Sphingomyelin and Niemann-Pick disease (NPD) type A and B
2. Therapy for type A and B NPD
3. Crystal structures of acid sphingomyelinase4. Summary

Summary• NPD type A and B present abnormal accumulation of sphingomyelin due to
deficient activity of ASM.
• NPD type A present progressive neurological symptoms.
• There are no approved therapies for ASMD except for enzyme replacement
therapy, which is effective only in NPD type B.
• Crystal structures of ASM revealed the mechanism of hydrolysis of sphingomyelin,in which two zinc ions activate a water molecule for nucleophilic attack ofphosphodiester bond.
• Saposin, proline-rich linker, catalytic and C-terminal domain together create acleft specific for sphingomyelin binding.