Structural divergence of GPI-80 in activated human neutrophils.
GPI-80 is a glycosylphosphatidylinositol (GPI)-anchored protein that is mainly expressed in human neutrophils. Previous studies using 3H9, a monoclonal antibody (mAb) against GPI-80, suggested that GPI-80 regulates leukocyte adherence and migration through Mac-1. GPI-80, which is anchored at the plasma membrane in resting neutrophils, moves into the pseudopodia and is released from activated human neutrophils. Here, we demonstrate that neutrophil activation affects GPI-80 dynamics using a new anti-GPI-80 mAb, designated 4D4, which is directed against the form of GPI-80 found on resting human neutrophils. Similar to 3H9, 4D4 influences Mac-1-dependent neutrophil adhesion. Treatment of purified GPI-80 with periodic acid and trypsin indicated that 3H9 and 4D4 recognize peptide and carbohydrate moieties, respectively. Stimulation with fMLP decreased the binding of 4D4 to GPI-80 on the neutrophil surface but increased the overall expression of GPI-80, as visualized by the 3H9 signal. Confocal laser microscopy revealed the 4D4 signal mainly on cell bodies and at a low level on pseudopodia during migration toward increasing concentrations of fMLP, whereas the 3H9 signal was observed in both areas. In addition, soluble GPI-80 released from activated neutrophils did not bind 4D4. These results suggest that there are two populations of GPI-80 that differ in the ability to bind 4D4. The 4D4-recognized form may regulate Mac-1-dependent neutrophil adhesion, and may subsequently be converted to a 4D4-unrecognized form during neutrophil activation.
Nitto
Takeaki
T
Department of Immunology and Parasitology, Yamagata University School of Medicine, Yamagata University, Yamagata, Japan.
Takeda
Yuji
Y
Yoshitake
Hiroshi
H
Sendo
Fujiro
F
Araki
Yoshihiko
Y
D000581:Amidohydrolases; D000818:Animals; D016466:CHO Cells; D002241:Carbohydrates / Q000737:chemistry; D002448:Cell Adhesion; D015815:Cell Adhesion Molecules / Q000737:chemistry*; D002462:Cell Membrane / Q000378:metabolism; D002465:Cell Movement; D006224:Cricetinae; D003412:Cricetulus; D058851:GPI-Linked Proteins; D017261:Glycosylphosphatidylinositols / Q000737:chemistry*; D006867:Hydrolases; D007962:Leukocytes / Q000378:metabolism; D051379:Mice; D009504:Neutrophils / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation
D002241:Carbohydrates; D015815:Cell Adhesion Molecules; D058851:GPI-Linked Proteins; D017261:Glycosylphosphatidylinositols; D006867:Hydrolases; D000581:Amidohydrolases; C121771:VNN2 protein, human