Proteomic analysis of plasma membrane and secretory vesicles from human neutrophils

Jethwaney Deepa, Islam, Leidal, De, Campbell, Nauseef, Gibson

Description

Polymorphonuclear neutrophils (PMN) constitute an essential cellular component of innate host defense against microbial invasion and exhibit a wide array of responses both to particulate and soluble stimuli. As the cells recruited earliest during acute inflammation, PMN respond rapidly and release a variety of potent cytotoxic agents within minutes of exposure to microbes or their products. PMN rely on the redistribution of functionally important proteins, from intracellular compartments to the plasma membrane and phagosome, as the means by which to respond quickly. To determine the range of membrane proteins available for rapid recruitment during PMN activation, we analyzed the proteins in subcellular fractions enriched for plasma membrane and secretory vesicles recovered from the light membrane fraction of resting PMN after Percoll gradient centrifugation and free-flow electrophoresis purification using mass spectrometry-based proteomics methods. Results To identify the proteins light membrane fractions enriched for plasma membrane vesicles and secretory vesicles, we employed a proteomic approach, first using MALDI-TOF (peptide mass fingerprinting) and then by HPLC-MS/MS using a 3D ion trap mass spectrometer to analyze the two vesicle populations from resting PMN. We identified several proteins that are functionally important but had not previously been recovered in PMN secretory vesicles. Two such proteins, 5-lipoxygenase-activating protein (FLAP) and dysferlin were further validated by immunoblot analysis. Conclusion Our data demonstrate the broad array of proteins present in secretory vesicles that provides the PMN with the capacity for remarkable and rapid reorganization of its plasma membrane after exposure to proinflammatory agents or stimuli.

Informations

• Publié par: biomed
• Publié le: 01 janvier 2007
• Nombre de lectures: 11
• Langue: English

Extrait

BioMed CentralProteome Science
Open AccessResearch
Proteomic analysis of plasma membrane and secretory vesicles
from human neutrophils
1 2 2Deepa Jethwaney , Md Rafiqul Islam , Kevin G Leidal , Daniel Beltran-
3 3 2Valero de Bernabe , Kevin P Campbell , William M Nauseef and
1Bradford W Gibson*
1 2Address: Buck Institute for Age Research, Novato, CA 94945, USA, Inflammation Program, Department of Medicine, University of Iowa and
3Veterans Administration Medical Center, Iowa City, IA 52240, USA and Howard Hughes Medical Institute, Senator Paul D. Wellstone Muscular
Dystrophy Cooperative Research Center, Department of Molecular Physiology and Biophysics, Department of Neurology, andDepartment of
Internal Medicine, University of Iowa, Iowa City, IA 52240, USA
Email: Deepa Jethwaney - [e-mail]; Md Rafiqul Islam - [e-mail]; Kevin G Leidal - [e-mail];
Daniel Beltran-Valero de Bernabe - [e-mail]; Kevin P Campbell - [e-mail]; William M Nauseef - william-
[e-mail]; Bradford W Gibson* - [e-mail]
* Corresponding author
Published: 10 August 2007 Received: 10 April 2007
Accepted: 10 August 2007
Proteome Science 2007, 5:12 doi:10.1186/1477-5956-5-12
This article is available from: http://www.proteomesci.com/content/5/1/12
© 2007 Jethwaney et al; licensee BioMed Central Ltd.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Abstract
Background: Polymorphonuclear neutrophils (PMN) constitute an essential cellular component
of innate host defense against microbial invasion and exhibit a wide array of responses both to
particulate and soluble stimuli. As the cells recruited earliest during acute inflammation, PMN
respond rapidly and release a variety of potent cytotoxic agents within minutes of exposure to
microbes or their products. PMN rely on the redistribution of functionally important proteins,
from intracellular compartments to the plasma membrane and phagosome, as the means by which
to respond quickly. To determine the range of membrane proteins available for rapid recruitment
during PMN activation, we analyzed the proteins in subcellular fractions enriched for plasma
membrane and secretory vesicles recovered from the light membrane fraction of resting PMN after
Percoll gradient centrifugation and free-flow electrophoresis purification using mass spectrometry-
based proteomics methods.
Results: To identify the proteins light membrane fractions enriched for plasma membrane vesicles
and secretory vesicles, we employed a proteomic approach, first using MALDI-TOF (peptide mass
fingerprinting) and then by HPLC-MS/MS using a 3D ion trap mass spectrometer to analyze the two
vesicle populations from resting PMN. We identified several proteins that are functionally
important but had not previously been recovered in PMN secretory vesicles. Two such proteins,
5-lipoxygenase-activating protein (FLAP) and dysferlin were further validated by immunoblot
analysis.
Conclusion: Our data demonstrate the broad array of proteins present in secretory vesicles that
provides the PMN with the capacity for remarkable and rapid reorganization of its plasma
membrane after exposure to proinflammatory agents or stimuli.
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partment contain a variety of functionally importantBackground
Human polymorphonuclear leukocytes (neutrophils or membrane proteins [reviewed in [14]]. During exposure
PMN) are essential for optimal host defense against to proinflammatory stimuli, the secretory vesicles readily
invading microorganisms and employ both oxygen- fuse with the plasma membrane, thereby integrating its
dependent and -independent agents in concert to kill and resident membrane proteins with those constitutively
degrade ingested microbe [1]. The cell biology of PMN is present at the PMN surface [14]. In this way the fusion of
especially tailored to mediate the rapid and efficient secretory vesicles with the plasma membrane transforms
responses that characterize the innate immune system the resting PMN to a cell more suited to deliver cytotoxic
early in inflammation. Stimulation of PMN triggers sev- agents against invading microbes or other threatening
eral concurrent events that together mount a potent cyto- noxious agents [15,16].
toxic response to invading microbes or other noxious
agents [2]. The purpose of the present study is to employ proteomic
analysis of plasma membrane and secretory vesicles from
The initiation of phagocytosis stimulates the assembly resting human PMN in order to define the repertoire of
and activation of the NADPH oxidase (reviewed in [3]), functionally important membrane proteins available in
resulting in the PMN undergoing a burst of oxygen con- secretory vesicles for rapid recruitment to the plasma
sumption and generation of reactive oxygen species. The membrane during PMN activation.
NADPH oxidase is a multicomponent enzyme complex
that is unassembled and inactive in the resting PMN, with Results
essential components segregated in distinct cellular com- Resolution of plasma membrane-enriched fractions from
partments (i.e. membrane vs. cytoplasm) in the unstimu- resting PMN
lated cell. When PMN are stimulated, the cytosolic The light membrane fraction recovered from a two-step
elements translocate to the plasma or phagosomal mem- Percoll density gradient separation of cavitated resting
brane where they associate with the membrane-bound fla- PMN [13], the γ fraction, is enriched for plasma mem-
vocytochrome b to form a functional oxidase complex. brane vesicles (PMV) but also contains secretory vesicles558
Simultaneously the intracellular granules fuse with the (SV), a labile intracellular compartment whose mem-
phagosomal membrane, thereby releasing their contents branes contain several functionally important proteins
into the same compartment as that in which the reactive [17]. In light of the lability of SV and the facility with
oxygen species are being generated [4-6]. The granule con- which they fuse with the plasma membrane, it was essen-
tents include proteolytic enzymes such as elastase [7] pro- tial to be confident that PMN used for study were truly at
teins that are directly toxic to target microbes such as the rest. In the absence of endotoxin contamination, PMN
defensins [8,9] or bactericidal permeability increasing isolated from heparinized venous blood using sequential
protein [10], and proteins that convert H O into more dextran sedimentation and differential density centrifuga-2 2
potent antimicrobial species [11]. Reactive oxygen spe- tion on Hypaque-Ficoll are neither primed nor stimu-
cies, antimicrobial proteins, and hydrolytic enzymes not lated: they do not consume oxygen, indicating that the
only act independently but also cooperate synergistically NADPH oxidase is neither assembled nor active, and their
to create an environment within the phagosome that is intracellular compartments remain intact [1]. For our
extremely inhospitable to the ingested microbe. Both oxi- studies, we routinely screen the status of NADPH oxidase
dase assembly and degranulation represent agonist- activity, using superoxide dismutase-inhibitable reduc-
dependent redistribution of prefabricated biological ele- tion of ferricytochrome C to quantitate oxidant produc-
ments, a strategy of cellular response that is especially tai- tion [18]. Routinely, PMN isolated by sequential dextran
lored to the physiologic responsibilities of PMN within sedimentation and differential density centrifugation on
the context of innate immunity and distinctly different Hypaque-Ficoll generate 1.01 ± 0.21 nmoles superoxide
6 from one dependent on transcriptional control of the pro- anion/10 PMN/10 min (n = 9), whereas PMN stimulated
duction of reactive molecules [12]. with 100 ng/ml of phorbol myristate acetate produce
6 78.47 ± 2.48 nmoles superoxide anion/10 PMN/10 min
Recent interest has focused on identification of the vari- (n = 9). Using the absence of oxidase activity as a criterion,
ous types of granules in PMN and their sequential mobi- PMN used in these studies were at rest. Another feature of
lization during activation. In addition to the distinct resting PMN is the presence of 85% of the flavocyto-
phox phox granule populations, PMN contain secretory vesicles, a chrome b (a heterodimer of gp91 and p22 and558
unique and easily mobilizable compartment that co-sedi- the membrane component of the phagocyte NADPH oxi-
ments with plasma membrane in the light membrane dase) in the specific granules [19]. To assess the distribu-
fraction of resting PMN [13]. Whereas the lumen of secre- tion of flavocytochrome b in PMN used in our studies,558
tory vesicles houses plasma proteins such as human we immunoblotted an equal number of cell equivalents
serum albumin, the membranes of this intracellular com- of specific granules, PMV, and SV, the subcellular com-
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phox partments in which gp91 is expressed [17]. Consistent
phox with previous reports, the m