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Isolation and characterization of complement receptor type 1 (CR1) storage
vesicles from human neutrophils using antibodies to the cytoplasmic tail of
CR1
A Kumar, E Wetzler and M Berger
Department of Pediatrics, Case Western Reserve University School of
Medicine, Cleveland, OH, USA.
Neutrophil (PMN) activation is associated with increased surface expression
of several membrane proteins that are translocated from intracellular
pools. Indirect evidence suggests that the intracellular storage pools of
complement receptor type 1 (CR1) in resting PMN are distinct from
traditional granules and may be the secretory vesicles in which albumin is
also stored, but it is not known if this compartment is homogeneous or
heterogeneous. To isolate and characterize the CR1- containing vesicles, we
used antibodies against unique sequences in the cytoplasmic tail of CR1.
Affinity-purified IgG was used to adsorb CR1 storage vesicles from the
light membrane fraction (gamma-band) of nitrogen cavitates of resting PMN.
The immunoadsorbent could quantitatively remove the CR1-containing
vesicles, whereas control adsorbents with nonimmune IgG showed no specific
binding of CR1. Immunoblots of specifically isolated vesicles also showed
enrichment of albumin, decay-accelerating factor, Fc gammaRIII, and CR3;
whereas HLA class I was not detectable in these vesicles. Enzyme assay of
specifically isolated vesicles after treatment with Triton X-100 showed
that these vesicles contained most of the cells' latent alkaline
phosphatase. An additional population of vesicles containing albumin, but
not CR1, and that did not bind to anti-CR1 adsorbent was also identified.
Immunoelectron microscopy showed that the specifically isolated vesicles
had mean diameters of 0.086 to 0.1 microm and stained positive for CR1 and
albumin. These results indicate that CR1 storage vesicles can be isolated
with antibodies against the cytoplasmic tail of CR1 and show that these
vesicles also contain albumin as well as glycosylphosphatidyl
inositol-anchored proteins. These results are most compatible with the
hypothesis that CR1-containing vesicles have arisen by endocytic retrieval
of proteins that had been on the plasma membrane.
Volume 89,
Issue 12,
pp. 4555-4565,
06/15/1997
Copyright © 1997 by The American Society of Hematology
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