Stabilization of granulocyte colony-stimulating factor and structurally analogous growth factors by anionic phospholipids.
Recombinant granulocyte colony-stimulating factor (rhG-CSF) interacts with liposomes composed of the anionic phospholipid dioleoylphosphatidylglycerol (DOPG), and this interaction enhances the stability of the protein [Collins, D., & Cha, Y. (1994) Biochemistry 33, 4521-4526]. In the present studies, we have examined the interaction of rhG-CSF with phospholipids other than DOPG. Fluorescence spectroscopy of rhG-CSF with a variety of lipid vesicles demonstrated that rhG-CSF inserts into bilayers of anionic, but not zwitterionic, phospholipids. Isothermal titration calorimetry of the interaction between DMPG and rhG-CSF indicates that the binding is saturable and involves 10 lipids/rhG-CSF. Studies of phosphatidylglycerols with varying alkyl chain lengths determined that the stabilization of rhG-CSF by anionic phospholipids required a certain alkyl chain length; no stabilization was observed with lipids of shorter chain length. Also investigated was the stabilization of other growth factors, which are structurally similar to rhG-CSF, by anionic phospholipids. These proteins include recombinant porcine somatotropin (rpSt), recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF), recombinant human interleukin 4 (rhIL-4), and recombinant human interleukin 2 (rhIL-2). The helical secondary structure of the proteins was recoverable after heating and cooling in the presence of anionic phospholipids as observed by circular dichroism; the presence of zwitterionic lipids did not induce this effect. Results of these investigations concluded that a group of structurally similar proteins interact preferentially with anionic phospholipids and that the complexation of the growth factors with vesicles composed of anionic phospholipids improves the stability of the proteins under conditions where they normally denature.
Rourke
A M
AM
Department of Pharmaceutics and Drug Delivery, Amgen, Inc., Thousand Oaks, California 91320, USA.
Cha
Y
Y
Collins
D
D
D002152:Calorimetry, Differential Scanning; D002942:Circular Dichroism; D016179:Granulocyte Colony-Stimulating Factor / Q000737:chemistry* / Q000378:metabolism; D006098:Granulocytes / Q000737:chemistry; D013006:Growth Hormone / Q000737:chemistry / Q000378:metabolism; D006133:Growth Substances / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D007378:Interleukins / Q000737:chemistry / Q000378:metabolism; D008081:Liposomes / Q000737:chemistry* / Q000378:metabolism; D010715:Phosphatidylglycerols / Q000737:chemistry / Q000378:metabolism; D010743:Phospholipids / Q000737:chemistry* / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013696:Temperature
D006133:Growth Substances; D007378:Interleukins; D008081:Liposomes; D010715:Phosphatidylglycerols; D010743:Phospholipids; D011994:Recombinant Proteins; D016179:Granulocyte Colony-Stimulating Factor; C051388:1,2-dioleoyl-sn-glycero-3-phosphoglycerol; D013006:Growth Hormone; C002773:dimyristoylphosphatidylglycerol