Skip to main content
SpringerLink
Log in
Book cover

Lipases and Phospholipases pp 201–225Cite as

Cloning of PLD2 from Baculovirus for Studies in Inflammatory Responses

  • Protocol
  • First Online:

Part of the book series: Methods in Molecular Biology ((MIMB,volume 861))

Abstract

The enzyme PLD hydrolyzes phosphodiester bonds of lipids in cell membranes. Phosphatidic acid, a chief product of PLD enzymatic activity, is a pleiotropic second messenger with key roles in membrane trafficking, cell invasion, cell growth, and anti-apoptosis. We describe in the present study molecular, cellular, and physiological methods to understand the mechanism of how the PLD2 isozyme regulates the process of inflammation. We describe here (1) a method that details phospholipase D2 (PLD2) cloning in the pBac expression vector, (2) the large-scale infection of Sf21 insect cells for protein production, (3) protein purification by TALON cobalt metal affinity matrix and subsequent assessment of PLD2 protein and lipase activity, (4) application of purified PLD2 protein for the study of Rac2 GTPase biology involving GTP binding by a pull-down assay and GTP/GDP exchange activity, (5) a method of PLD2 expression that involves mammalian cells, (6) a physiological application as relates to adhesion, chemotaxis, and phagocytosis, and (7) a model that integrates the results of a PLD–GTPase interaction from the molecular to the physiological contexts.

This is a preview of subscription content, log in via an institution.

Protocol
USD   49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD   139.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD   179.00
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD   219.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Learn about institutional subscriptions

Springer Nature is developing a new tool to find and evaluate Protocols. Learn more

References

  1. Frohman MA, Sung TC, Morris AJ (1999) Mammalian phospholipase D structure and regulation. Biochim Biophys Acta 1439:175–186

    PubMed  CAS  Google Scholar 

  2. Gomez-Cambronero J (2010) New concepts in phospholipase D signaling in inflammation and cancer. SciWorldJ 10:1356–1369

    CAS  Google Scholar 

  3. Exton JH (2000) Phospholipase D. Ann N Y Acad Sci 905:61–68

    Article  PubMed  CAS  Google Scholar 

  4. Allgyer TT, Wells MA (1979) Phospholipase D from savoy cabbage: purification and preliminary kinetic characterization. Biochemistry 18:5348–5353

    Article  PubMed  CAS  Google Scholar 

  5. Wang X, Xu L, Zheng L (1994) Cloning and expression of phosphatidylcholine-hydrolyzing phospholipase D from Ricinus communis L. J Biol Chem 269:20312–20317

    PubMed  CAS  Google Scholar 

  6. Ueki J, Morioka S, Komari T et al (1995) Purification and characterization of phospholipase D (PLD) from rice (Oryza sativa L.) and cloning of cDNA for PLD from rice and maize (Zea mays L.). Plant Cell Physiol 36:903–914

    PubMed  CAS  Google Scholar 

  7. Salak J, Roch P, Soucek A (1979) Isolation of corynebacterial enzymes from cultivation media by gel filtration and ion-exchange chromatography. Toxicon 17:655–658

    Article  PubMed  CAS  Google Scholar 

  8. Rudge SA, Engebrecht J (1999) Regulation and function of PLDs in yeast. Biochim Biophys Acta 1439:167–174

    PubMed  CAS  Google Scholar 

  9. Colley WC, Altshuller YM, Sue-Ling CK et al (1997) Cloning and expression analysis of murine phospholipase D1. Biochem J 326(Pt 3):745–753

    PubMed  CAS  Google Scholar 

  10. Min DS, Park SK, Exton JH (1998) Characterization of a rat brain phospholipase D isozyme. J Biol Chem 273:7044–7051

    Article  PubMed  CAS  Google Scholar 

  11. Lopez I, Arnold RS, Lambeth JD (1998) Cloning and initial characterization of a human phospholipase D2 (hPLD2). ADP-ribosylation factor regulates hPLD2. J Biol Chem 273:12846–12852

    Article  PubMed  CAS  Google Scholar 

  12. Park SH, Chun YH, Ryu SH et al (1998) Assignment of human PLD1 to human chromosome band 3q26 by fluorescence in situ hybridization. Cytogenet Cell Genet 82:224

    Article  PubMed  CAS  Google Scholar 

  13. Katayama K, Kodaki T, Nagamachi Y et al (1998) Cloning, differential regulation and tissue distribution of alternatively spliced isoforms of ADP-ribosylation-factor-dependent phospholipase D from rat liver. Biochem J 329:647–652

    PubMed  CAS  Google Scholar 

  14. Hammond SM, Jenco JM, Nakashima S et al (1997) Characterization of two alternately spliced forms of phospholipase D1. Activation of the purified enzymes by phosphatidylinositol 4,5-bisphosphate, ADP-ribosylation factor, and Rho family monomeric GTP-binding proteins and protein kinase C-alpha. J Biol Chem 272:3860–3868

    Article  PubMed  CAS  Google Scholar 

  15. Park SH, Ryu SH, Suh PG et al (1998) Assignment of human PLD2 to chromosome band 17p13.1 by fluorescence in situ hybridization. Cytogenet Cell Genet 82:225–225

    Article  PubMed  CAS  Google Scholar 

  16. Steed PM, Clark KL, Boyar WC et al (1998) Characterization of human PLD2 and the analysis of PLD isoform splice variants. FASEB J 12:1309–1317

    PubMed  CAS  Google Scholar 

  17. Brown HA, Henage LG, Preininger AM et al (2007) Biochemical analysis of phospholipase D. Methods Enzymol 434:49–87

    Article  PubMed  CAS  Google Scholar 

  18. Hink WF, Thomsen DR, Davidson DJ et al (1991) Expression of three recombinant proteins using baculovirus vectors in 23 insect cell lines. Biotechnol Prog 7:9–14

    Article  PubMed  CAS  Google Scholar 

  19. Vaughn JL, Goodwin RH, Tompkins GJ et al (1977) The establishment of two cell lines from the insect Spodoptera frugiperda (Lepidoptera; Noctuidae). In Vitro 13:213–217

    Article  PubMed  CAS  Google Scholar 

  20. Kusner DJ, Hall CF, Jackson S (1999) Fc gamma receptor-mediated activation of phospholipase D regulates macrophage phagocytosis of IgG-opsonized particles. J Immunol 162:2266–2274

    PubMed  CAS  Google Scholar 

  21. Lehman N, Di Fulvio M, McCray N et al (2006) Phagocyte cell migration is mediated by phospholipases PLD1 and PLD2. Blood 108:3564–3572

    Article  PubMed  CAS  Google Scholar 

  22. Raymond FD, Fortunato G, Moss DW et al (1994) Inositol-specific phospholipase D activity in health and disease. Clin Sci (Lond) 86:447–451

    CAS  Google Scholar 

  23. Cha SH, Lee WK, Kim KA et al (1998) Effect of silica on phospholipase D activity in rat alveolar macrophages. Ind Health 36:258–262

    Article  PubMed  CAS  Google Scholar 

  24. Laine J, Bourgoin S, Bourassa J et al (2000) Subcellular distribution and characterization of rat pancreatic phospholipase D isoforms. Pancreas 20:323–336

    Article  PubMed  CAS  Google Scholar 

  25. Di Fulvio M, Gomez-Cambronero J (2005) Phospholipase D (PLD) gene expression in human neutrophils and HL-60 differentiation. J Leukoc Biol 77:999–1007

    Article  PubMed  Google Scholar 

  26. Liscovitch M, Czarny M, Fiucci G et al (2000) Phospholipase D: molecular and cell biology of a novel gene family. Biochem J 345(Pt 3):401–415

    Article  PubMed  CAS  Google Scholar 

  27. Walker SJ, Brown HA (2002) Specificity of Rho insert-mediated activation of phospholipase D1. J Biol Chem 277:26260–26267

    Article  PubMed  CAS  Google Scholar 

  28. Hart MJ, Eva A, Evans T et al (1991) Catalysis of guanine nucleotide exchange on the CDC42Hs protein by the dbl oncogene product. Nature 354:311–314

    Article  PubMed  CAS  Google Scholar 

  29. Peng HJ, Henkels KM, Mahankali M et al (2011) Evidence for two CRIB domains in phospholipase D2 (PLD2) that the enzyme uses to specifically bind to the small GTPase Rac2. J Biol Chem 286:16308–16320

    Google Scholar 

  30. Peng HJ, Henkels KM, Mahankali M et al (2011) The dual effect of Rac2 on PLD2 regulation that explains both onset and termination of chemotaxis. Mol Cell Biol 31:2227–2240

    Article  PubMed  CAS  Google Scholar 

  31. Powner DJ, Payne RM, Pettitt TR et al (2005) Phospholipase D2 stimulates integrin-­mediated adhesion via phosphatidylinositol 4-phosphate 5-kinase Igamma b. J Cell Sci 118:2975–2986

    Article  PubMed  CAS  Google Scholar 

  32. Knoepp SM, Chahal MS, Xie Y et al (2008) Effects of active and inactive phospholipase D2 on signal transduction, adhesion, migration, invasion, and metastasis in EL4 lymphoma cells. Mol Pharmacol 74:574–584

    Article  PubMed  CAS  Google Scholar 

  33. Frondorf K, Henkels KM, Frohman MA et al (2010) Phosphatidic acid (PA) is a leukocyte chemoattractant that acts through S6 kinase signaling. J Biol Chem 285:15837–15847

    Article  PubMed  CAS  Google Scholar 

  34. Iyer SS, Barton JA, Bourgoin S et al (2004) Phospholipases D1 and D2 coordinately regulate macrophage phagocytosis. J Immunol 173:2615–2623

    PubMed  CAS  Google Scholar 

  35. Corrotte M, Chasserot-Golaz S, Huang P et al (2006) Dynamics and function of phospholipase D and phosphatidic acid during phagocytosis. Traffic 7:365–377

    Article  PubMed  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Biochemistry and Molecular Biology, Wright State University School of Medicine, Dayton, OH, USA

    Julian Gomez-Cambronero & Karen M. Henkels

Authors
  1. Julian Gomez-Cambronero
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Karen M. Henkels
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Julian Gomez-Cambronero .

Editor information

Editors and Affiliations

  1. CIATEJ-CONACYT, Industrial Biotechnology Unit, Avda. Normalistas 800, Guadalajara, 44270, Jalisco, Mexico

    Georgina Sandoval

Rights and permissions

Reprints and permissions

Copyright information

© 2012 Springer Science+Business Media New York

About this protocol

Cite this protocol

Gomez-Cambronero, J., Henkels, K.M. (2012). Cloning of PLD2 from Baculovirus for Studies in Inflammatory Responses. In: Sandoval, G. (eds) Lipases and Phospholipases. Methods in Molecular Biology, vol 861. Humana Press. https://doi.org/10.1007/978-1-61779-600-5_13

Download citation

  • DOI: https://doi.org/10.1007/978-1-61779-600-5_13

  • Published:

  • Publisher Name: Humana Press

  • Print ISBN: 978-1-61779-599-2

  • Online ISBN: 978-1-61779-600-5

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation