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Differences in Stability, Activity and Mutation Effects Between Human and Mouse Leucine-Rich Repeat Kinase 2

  • Rebekah G. Langston
  • Iakov N. Rudenko
  • Ravindran Kumaran
  • David N. Hauser
  • Alice Kaganovich
  • Luis Bonet Ponce
  • Adamantios Mamais
  • Kelechi Ndukwe
  • Allissa A. Dillman
  • Amr M. Al-Saif
  • Aleksandra Beilina
  • Mark R. Cookson
Original Paper

Abstract

Mutations in the Leucine-rich repeat kinase 2 (LRRK2) gene have been implicated in the pathogenesis of Parkinson’s disease (PD). Identification of PD-associated LRRK2 mutations has led to the development of novel animal models, primarily in mice. However, the characteristics of human LRRK2 and mouse Lrrk2 protein have not previously been directly compared. Here we show that proteins from different species have different biochemical properties, with the mouse protein being more stable but having significantly lower kinase activity compared to the human orthologue. In examining the effects of PD-associated mutations and risk factors on protein function, we found that conserved substitutions such as G2019S affect human and mouse LRRK2 proteins similarly, but variation around position 2385, which is not fully conserved between humans and mice, induces divergent in vitro behavior. Overall our results indicate that structural differences between human and mouse LRRK2 are likely responsible for the different properties we have observed for these two species of LRRK2 protein. These results have implications for disease modelling of LRRK2 mutations in mice and on the testing of pharmacological therapies in animals.

Keywords

Parkinson’s disease Kinase activity Protein stability Level of expression Mouse model 

Notes

Acknowledgements

We are grateful to Dr. Matthew S. Goldberg (University of Texas Southwestern Medical Center, USA) for sharing a mouse Lrrk2 cDNA construct. This work was supported by the Intramural Research Program of the National Institute on Aging, NIH, by The Michael J. Fox Foundation for Parkinson’s Research, and by a Parkinson’s Foundation- American Parkinson Disease Association Summer Student Fellowship, PF-APDA-SFW-1742.

Supplementary material

11064_2018_2650_MOESM1_ESM.pdf (251 kb)
Supplementary figure S1. Higher mouse LRRK2 protein expression is not explained by increased protein stability. A. HEK293FT cells were transfected with Flag-tagged human or mouse LRRK2 and subjected to a 35S-cysteine/35S-methionine “pulse” followed by a “chase” with media enriched with “cold” cysteine and methionine. Cell lysates were subjected to immunoprecipitation (IP) for Flag and exposed to a storage phosphor screen (upper panel), then blotted for Flag (lower panel). The representative blots shown are taken from an experiment in which cells were collected at 32 hours. In a separate experiment cells were collected at 2 hours, and not at 32 hours. B. Quantification of 35S-LRRK2 relative to Flag-LRRK2 for human and mouse LRRK2 from n=3 independent experiments, with technical n=3 for each construct at each time point. Error bars indicate SEM. The best-fit lines shown are semilog lines, where X is linear, and Y is log. A one phase decay non-linear regression equation was used to calculate half-life of each protein (Human LRRK2: half-life = 3.83h, 95%CI=2.71-5.68, R2=0.84. Mouse Lrrk2: half-life = 3.36h, 95%CI=2.60-4.38, R2=0.904). (PDF 252 KB)
11064_2018_2650_MOESM2_ESM.pdf (549 kb)
Supplementary figure S2. Mouse LRRK2 is expressed at higher levels than human protein in mouse cells. A-C. Primary mouse glial cells (A), N2a cells (B) or NIH 3t3 cells (C) were transfected with Flag-tagged human or mouse LRRK2, or mock transfected and protein levels measured by western blot using a flag antibody. Cyclophilin B is used as a loading control for each lane. (PDF 550 KB)
11064_2018_2650_MOESM3_ESM.pdf (104 kb)
Supplementary figure S3. Mouse and human LRRK2 self-interact at similar levels. A. HEK293FT cells were co-transfected with GFP-tagged LRRK2 and indicated Flag-tagged human or mouse LRRK2 constructs, with GUS as a negative control. Cell lysates were subjected to immunoprecipitation (IP) for GFP and blotted for GFP (upper panel) and Flag (lower panels). Inputs for the IP are shown on the left. B. Quantification of IP for Flag-tagged proteins relative to inputs for indicated human and mouse constructs from n=3 independent experiments. Error bars indicate SEM, **, p < 0.01; ns, non-significant by Tukey’s post-hoc test from one way ANOVA compared to WT human LRRK2. (PDF 104 KB)
11064_2018_2650_MOESM4_ESM.pdf (58 kb)
Supplementary figure S4. Enzyme activity of mouse and human LRRK2 estimated using model peptide, Nictide. Quantification of n=3 independent experiments using indicated constructs of the phosphorylation of the Nictide peptide. *, p < 0.05; ***, p < 0.001; ns, non-significant by Tukey’s post-hoc test compared to WT human LRRK2 from one way ANOVA (F10,55=20.87, p < 0.001, n=6 samples per construct). (PDF 58 KB)

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Copyright information

© This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply 2018

Authors and Affiliations

  • Rebekah G. Langston
    • 1
  • Iakov N. Rudenko
    • 1
    • 2
  • Ravindran Kumaran
    • 1
  • David N. Hauser
    • 1
    • 5
  • Alice Kaganovich
    • 1
  • Luis Bonet Ponce
    • 1
  • Adamantios Mamais
    • 1
  • Kelechi Ndukwe
    • 1
    • 3
  • Allissa A. Dillman
    • 1
    • 4
  • Amr M. Al-Saif
    • 1
  • Aleksandra Beilina
    • 1
  • Mark R. Cookson
    • 1
    • 6
  1. 1.Laboratory of Neurogenetics, Cell Biology and Gene Expression SectionNIA, NIHBethesdaUSA
  2. 2.Department of Neurology, SUNY at Stony BrookHealth Science CenterStony BrookUSA
  3. 3.Medical College of Wisconsin, Medical SchoolMilwaukeeUSA
  4. 4.Laboratory of Receptor Biology and Gene Expression, Center for Cancer ResearchNational Cancer InstituteBethesdaUSA
  5. 5.Sanford Burnham Prebys Medicial Discovery InstituteLa JollaUSA
  6. 6.Laboratory of Neurogenetics, Cell Biology and Gene Expression SectionNational Institute on Aging, NIHBethesdaUSA

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