Lymphocyte phosphatase-associated phosphoprotein (LPAP) as CD45 protein stability regulator

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Abstract

Lymphocyte phosphatase-associated phosphoprotein (LPAP) is a protein of unknown function. Its close interaction with CD45 phosphatase suggests that LPAP may potentially regulate CD45, but direct biochemical evidence for this has not yet been obtained. We found that on Jurkat lymphoid cells the levels of LPAP and CD45 proteins are interrelated and well correlated with each other. Knockout of LPAP leads to a decrease, and its overexpression, on the contrary, causes an increase in the surface expression of CD45. No such correlation is found in non-lymphoid K562 cells. In the absence of LPAP, upon activation of Jurkat cells, a decrease in the expression of the activation marker CD69 was observed. This may be due to both direct and indirect effects of LPAP. We have hypothesized that LPAP is a regulator of the expression level of CD45 phosphatase.

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N. А. Kruglova

Institute of Gene Biology, Russian Academy of Sciences

Author for correspondence.
Email: natalya.a.kruglova@yandex.ru

Center for Precision Genome Editing and Genetic Technologies for Biomedicine

Russian Federation, 119334, Moscow

D. V. Mazurov

National Research Center Institute of Immunology, Federal Medical Biological Agency of Russia

Email: natalya.a.kruglova@yandex.ru
Russian Federation, 115522, Moscow

A. V. Filatov

National Research Center Institute of Immunology, Federal Medical Biological Agency of Russia; Lomonosov Moscow State University

Email: avfilat@yandex.ru

Department of Immunology, Faculty of Biology

Russian Federation, 115522, Moscow; 119234, Moscow

References

  1. Schraven, B., Schoenhaut, D., Bruyns, E., Koretzky, G., Eckerskorn, C., et al. (1994) LPAP, a novel 32-kDa phosphoprotein that interacts with CD45 in human lymphocytes, J. Biol. Chem., 269, 29102-29111.
  2. Rheinländer, A., Schraven, B., and Bommhardt, U. (2018) CD45 in human physiology and clinical medicine, Immunol. Lett., 196, 22-32, https://doi.org/10.1016/j.imlet.2018.01.009.
  3. Kleiman, E., Salyakina, D., De Heusch, M., Hoek, K. L., Llanes, J. M., et al. (2015) Distinct transcriptomic features are associated with transitional and mature B-cell populations in the mouse spleen, Front. Immunol., 6, 30, https:// doi.org/10.3389/fimmu.2015.00030.
  4. Kruglova, N. A., Meshkova, T. D., Kopylov, A. T., Mazurov, D. V., and Filatov, A. V. (2017) Constitutive and activation-dependent phosphorylation of lymphocyte phosphatase-associated phosphoprotein (LPAP), PLoS One, 12, e0182468, https://doi.org/10.1371/journal.pone.0182468.
  5. Kruglova, N., Filatov, A. (2019) T cell receptor signaling results in ERK-dependent Ser163 phosphorylation of lymphocyte phosphatase-associated phosphoprotein, Biochem. Biophys. Res. Commun., 519, 559-565, https://doi.org/ 10.1016/j.bbrc.2019.09.041.
  6. Takeda, A., Matsuda, A., Paul, R. M. J., and Yaseen, N. R. (2004) CD45-associated protein inhibits CD45 dimerization and up-regulates its protein tyrosine phosphatase activity, Blood, 103, 3440-3447, https://doi.org/10.1182/blood-2003-06-2083.
  7. Schraven, B., Kirchgessner, H., Gaber, B., Samstag, Y., and Meuer, S. (1991) A functional complex is formed in human T lymphocytes between the protein tyrosine phosphatase CD45, the protein tyrosine kinase p56lck and pp32, a possible common substrate, Eur. J. Immunol., 21, 2469-2477, https://doi.org/10.1002/eji.1830211025.
  8. Filatov, A., Kruglova, N., Meshkova, T., and Mazurov, D. (2015) Lymphocyte phosphatase-associated phosphoprotein proteoforms analyzed using monoclonal antibodies, Clin. Transl. Immunol., 4, e44, https://doi.org/10.1038/cti.2015.22.
  9. Ding, I., Bruyns, E., Li, P., Magada, D., Paskind, M., et al. (1999) Biochemical and functional analysis of mice deficient in expression of the CD45-associated phosphoprotein LPAP, Eur. J. Immunol., 29, 3956-3961, https://doi.org/ 10.1002/(SICI)1521-4141(199912)29:12<3956::AID-IMMU3956>3.0. CO;2-G.
  10. Matsuda, A., Motoya, S., Kimura, S., McInnis, R., Maizel, A. L., et al. (1998) Disruption of lymphocyte function and signaling in CD45-associated protein-null mice, J. Exp. Med., 187, 1863-1870, https://doi.org/10.1084/jem.187.11.1863.
  11. Kung, C., Okumura, M., Seavitt, J. R., Noll, M. E., White, L. S., et al. (1999) CD45-associated protein is not essential for the regulation of antigen receptor-mediated signal transduction, Eur. J. Immunol., 29, 3951-3955, https:// doi.org/10.1002/(SICI)1521-4141(199912)29:12<3951::AID-IMMU3951>3.0. CO;2-9.
  12. Bruyns, E., Kirchgessner, H., Meuer, S., Schraven, B. (1998) Biochemical analysis of the CD45-p56(Lck) complex in Jurkat T cells lacking expression of lymphocyte phosphatase-associated phosphoprotein, Int. Immunol., 10, 185-194, https://doi.org/10.1093/intimm/10.2.185.
  13. Kitamura, K., Matsuda, A., Motoya, S., Takeda, A. (1997) CD45-associated protein is a lymphocyte-specific membrane protein expressed in two distinct forms, Eur. J. Immunol., 27, 383-388, https://doi.org/10.1002/eji.1830270207.
  14. Hsu, P. D., Scott, D. A., Weinstein, J. A., Ran, F. A., Konermann, S., Agarwala, V., et al. (2013) DNA targeting specificity of RNA-guided Cas9 nucleases, Nat. Biotechnol., 31, 827-832, https://doi.org/10.1038/nbt.2647.
  15. Mali, P., Yang, L., Esvelt, K. M., Aach, J., Guell, M., et al. (2013) RNA-guided human genome engineering via Cas9, Science, 339, 823-826, https://doi.org/10.1126/science.1232033.
  16. Ran, F. A., Hsu, P. D., Lin, C.-Y., Gootenberg, J. S., Konermann, S., et al. (2013) Double nicking by RNA-guided CRISPR Cas9 for enhanced genome editing specificity, Cell, 154, 1380-1389, https://doi.org/10.1016/j.cell.2013.08.021.
  17. Zotova, A., Pichugin, A., Atemasova, A., Knyazhanskaya, E., Lopatukhina, E., et al. (2019) Isolation of gene-edited cells via knock-in of short glycophosphatidylinositol-anchored epitope tags, Sci. Rep., 9, 3132, https:// doi.org/10.1038/s41598-019-40219-z.
  18. Мазуров Д. В. (2020) В кн. Методы редактирования генов и геномов (под pед. Закияна С. М., Медведева С. П., Дементьевой Е. В., Власова В. В.) Издательство СО РАН, Новосибирск, с. 413-444.
  19. Zotova, A., Lopatukhina, E., Filatov, A., Khaitov, M., and Mazurov, D. (2017) Gene editing in human lymphoid cells: role for donor DNA, type of genomic nuclease and cell selection method, Viruses, 9, 325, https://doi.org/10.3390/v9110325.
  20. Koretzky, G. A., Picus, J., Schultz, T., and Weiss, A. (1991) Tyrosine phosphatase CD45 is required for T-cell antigen receptor and CD2-mediated activation of a protein tyrosine kinase and interleukin 2 production, Proc. Natl. Acad. Sci. USA, 88, 2037-2041, https://doi.org/10.1073/pnas.88.6.2037.
  21. Leitenberg, D., Falahati, R., Lu, D. D., and Takeda, A. (2007) CD45-associated protein promotes the response of primary CD4 T cells to low-potency T-cell receptor (TCR) stimulation and facilitates CD45 association with CD3/TCR and lck, Immunology, 121, 545-554, https://doi.org/10.1111/j.1365-2567.2007.02602.x.
  22. Gaud, G., Lesourne, R., and Love, P. E. (2018) Regulatory mechanisms in T cell receptor signalling, Nat. Rev. Immunol., 18, 485-497, https://doi.org/10.1038/s41577-018-0020-8.
  23. Burr, M. L., Sparbier, C. E., Chan, Y.-C., Williamson, J. C., Woods, K., et al. (2017) CMTM6 maintains the expression of PD-L1 and regulates anti-tumour immunity, Nature, 549, 101-105, https://doi.org/10.1038/nature23643.
  24. Oates, M. E., Romero, P., Ishida, T., Ghalwash, M., Mizianty, M. J., et al. (2012) D2P2: database of disordered protein predictions, Nucleic Acids Res., 41, D508-D516, https://doi.org/10.1093/nar/gks1226.
  25. Uversky, V. N. (2019) Intrinsically disordered proteins and their “mysterious” (meta)physics, Front. Phys., 7, https://doi.org/10.3389/fphy.2019.00010.
  26. Wright, P. E., Dyson, H. J. (2015) Intrinsically disordered proteins in cellular signalling and regulation, Nat. Rev. Mol. Cell Biol., 16, 18-29, https://doi.org/10.1038/nrm3920.
  27. Marchetti, P., Antonov, A., Anemona, L., Vangapandou, C., Montanaro, M., et al. (2021) New immunological potential markers for triple negative breast cancer: IL18R1, CD53, TRIM, Jaw1, LTB, PTPRCAP, Discov. Oncol., 12, 6, https://doi.org/10.1007/s12672-021-00401-0.

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2. Fig. 1. The level of LPAP and CD45 proteins decreases in the absence of a partner protein. a – Representative cytograms of LPAP expression on Jurkat wild–type (WT) cells, as well as on CD45KO or LPAPKO cells; b – LPAP expression according to the results of testing 7 CD45KO clones; c, d - expression of CD45 and "extraneous" CD59 and CD98 proteins in Jurkat LPAPmKO (c) or LPAPpKO (dThe expression levels of LPAP, CD45, CD59 and CD98 proteins were determined using flow cytometry. The normalized CD45 expression level was compared with a control value of 100 using a single-sample Student t-test (b). To compare the protein levels in wild-type and knockout cells, the ANOVA method was used with post hoc analysis using the Tukey test (b, d), **** p < 0.0001

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3. Figure 2. CD45 level correlates with LPAP protein level. a, b – Correlation between LPAP and CD45 (a) or LPAP and CD98 (b) levels in Jurkat LPAPKO clones with LPAPWT reexpression; c – correlation between LPAP and CD45 levels in Jurkat LPAPWT clones; d, e – LPAP (d) and CD45 (e) levels on Jurkat LPAPpKO cells, stably transduced with an increasing dose of the virus for LPAPWT reexpression (#1, #2, #3) or GFP expression. LPAP expression is normalized relative to Jurkat LPAPWT cells. For CD45, the values for the surface (sCD45) and total (tCD45) levels are shown; e – correlation between LPAP and CD45 levels in Jurkat LPAPKO clones stably transfected by LPAPWT. Expression levels were determined by MFI values, which were normalized relative to the average value for Jurkat WT after subtracting the background level in Jurkat CD45KO or Jurkat LPAPKO cells. Individual values are given, as well as the average ± SD. The normalized level of LPAP expression in the transduced cells was compared with a value of zero in cells expressing GFP using a single-sample Student t-test (g). To compare the levels of surface (s) and total (t) CD45 in Jurkat LPAPpKO cells and transducers, the ANOVA method with post hoc analysis using the Tukey test was used. *** p < 0,001, **** p < 0,0001. The average values for the LPAP and sCD45 levels from panel D were used to calculate the correlation (e)

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4. Fig. 3. CD45 levels increase in cells with increased LPAP expression. a, b – LPAP protein levels were analyzed in Jurkat and Jurkat LPAP-Flag cell lines after the first and second sorting (sort 1 and sort 2). Cells were lysed (a, b), LPAP or CD45 protein was isolated using immunoprecipitation (IP) (b), samples were separated by electrophoresis in 12% (a) or 18% (b) gel, and Western blotting with these antibodies was performed. The lower bolt (b) was stained with antibodies against Flag and LPAP. The LPAP and LPAP-Flag forms are indicated by arrows; b, g – expression of LPAP and CD45 on the surface of wild-type K562 cells, as well as stably transduced LPAP-Flag

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5. Fig. 4. Comparison of the expression of CD69 and CD3 molecules on wild-type Jurkat cells and with LPAP knockout. a – Expression of CD69 on cells activated by PMA; b – expression of CD69 on cells activated by the OKT3 antibody; c – expression of CD3 on cells activated by the OKT3 antibody. The levels of CD69 (b) and CD3 (c) were compared using a two-sample t-test. * p < 0,05, ** p < 0,001, **** p < 0,0001.

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