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Blood, 1 May 2003, Vol. 101, No. 9, pp. 3751-3752
CORRESPONDENCE
To the editor:
New and old integral proteins of the human erythrocyte membrane
Salzer et al, from the University of Vienna, have recently
described that vesicles released from Ca++/Ca++
ionophore-treated erythrocytes are enriched in lipids and proteins that
are typically found within lipid microdomains of the parent cell's
plasma membrane, the so-called "lipid rafts."1 The
microvesicles shed from Ca++-loaded erythrocytes, processed
for the separation of lipid domains, appear to contain the conventional
markers of lipid rafts, cholesterol and ganglioside
GM1.1 The novelty of the data is in the large number of membrane-associated proteins, most of which were previously undetected, found by Salzer et al in the human erythrocytes: stomatin, flotillin-1, flotillin-2, synexin, and sorcin. These proteins appear to
be associated with lipid rafts, and, most important, some of them are
enriched in the vesicles, relative to the parent cell's membrane. They
share this property with the family of exofacial proteins inserted in
the membrane via a glycosyl phosphatidylinositol (GPI) anchor. In
erythrocytes, the most famous member of this family is
acetylcholinesterase (AChE), whose enrichment in
Ca++-dependent vesicles was known well before the structure
of the GPI anchor was elucidated.2,3 If one takes the amount of membrane phospholipids as a measure of
membrane surface extension (as is common and correct practice) and then
normalizes the amount of AChE, as enzymatic activity, over membrane
phospholipids, a 3- to 4-fold enrichment of AChE in microvesicles, with
respect to erythrocytes, is observed. This figure, supported by several
independent reports in the past,3 was easily confirmed in
our lab. For reasons that were not explicitly stated in their paper,
and that therefore remain obscure to the reader, Salzer et al chose to
normalize AChE activity to hemoglobin content of vesicles and cells,
and were able to calculate a different figure. Thus, the amount of AChE
in vesicles, when referred to hemoglobin, is "roughly 80 times" the
amount in the parent cells.1 This figure may seem
confusing for a reader more accustomed to the old notion of a 3- to
4-fold enrichment. However, we must say that this result is correct. A
185-nm spherical vesicle2 has a volume of approximately
0.0033 µm3, and a surface area of approximately
0.108 µm2. The surface-to-volume ratio (S/V) is
therefore approximately 33 µm 1. The measured S/V of
erythrocytes4 (total population of cells) is approximately
1.59 µm1. Thus, the S/V in vesicles is roughly 20 times
the S/V in erythrocytes. A 3- to 4-fold enrichment in AChE in vesicles,
calculated by normalization of AChE activity over membrane surface
extension (phospholipids), becomes a 60- to 80-fold enrichment when
normalizing to cell volume (hemoglobin): a good fit. However,
it was probably not necessary to introduce a new procedure for
quantifying AChE enrichment in vesicles, since it does not constitute
an original finding. However, Salzer et al go one step further and
claim that band 3 protein and glycophorins are strongly decreased in
vesicles compared with cells. The way they demonstrate this is clearly
incorrect: sodium dodecyl sulfate-polyacrylamide gel
electrophoresis gels were loaded with equivalent amounts of
erythrocyte membranes and vesicle membranes by taking AChE as the
normalizing parameter. Then, band 3 was stained (glycophorins were not,
however) and shown to be decreased in vesicles.1(Fig 2) But
since AChE is enriched in the membrane of vesicles with respect to
cells, this method will always detect a decrease of anything that would
otherwise keep constant in the membrane of vesicles. Therefore, we
would prefer to adhere to the well-documented notion that band 3 is
equally represented, per unit surface area, in vesicles and
cells,3 as we have found the same result by direct measurements of properly loaded gels in our lab. Salzer et al conclude
that, "interestingly, only trace amounts of the flotillins are found in the vesicles [italics added]."1 Maybe
flotillins are indeed present in more than trace amounts in vesicles.
It is only a matter of watching more carefully.
Giampaolo Minetti and Annarita Ciana
Correspondence: Giampaolo Minetti, Universita' di Pavia,
Dipartimento di Biochimica, via Bassi 21 - 27100 Pavia,
Italy; e-mail: minetti{at}unipv.it
References
1.
Salzer U, Hinterdorfer P, Hunger U, Borker C, Prohaska R.
Ca++-dependent vesicle release from erythrocytes involves stomatin-specific lipid rafts, synexin (annexin VII), and sorcin.
Blood.
2002;99:2569-2577[Abstract/Free Full Text].
2.
Lutz HU, Liu SC, Palek J.
Release of spectrin-free vesicles from human erythrocytes during ATP depletion: I. Characterization of spectrin-free vesicles.
J Cell Biol.
1977;73:548-560[Abstract/Free Full Text].
3.
Butikofer P, Kuypers FA, Xu CM, Chiu DT, Lubin B.
Enrichment of two glycosyl-phosphatidylinositol-anchored proteins, acetylcholinesterase and decay accelerating factor, in vesicles released from human red blood cells.
Blood.
1989;74:1481-1485[Abstract/Free Full Text].
4.
Waugh RE, Narla M, Jackson CW, Mueller TJ, Suzuki T, Dale GL.
Rheologic properties of senescent erythrocytes: loss of surface area and volume with red blood cell age.
Blood.
1992;79:1351-1358[Abstract/Free Full Text].
Response:
Segregation of lipid raft proteins during calcium-induced
vesiculation of erythrocytes
The erythrocyte membrane contains lipid rafts,1-3
with stomatin, the flotillins, and glycosylphosphatidyl-inositol
(GPI)-linked proteins as the major integral proteins, whereas band 3 protein and the glycophorins are largely absent. As microvesicles
released from erythrocytes after Ca++ treatment are
specifically enriched in the GPI-linked enzyme acetylcholinesterase
(AChE),4 it is likely that lipid rafts are
involved in the vesiculation process. We showed that microvesicles in
fact contain lipid rafts; however, the relative amounts of the raft
proteins differ between microvesicles and the erythrocyte membrane.5 These results suggest a calcium-induced
segregation of different types of lipid rafts, with stomatin-specific
rafts enriched in microvesicles and flotillins depleted. The letter of Minetti and Ciana refers to a paragraph in "Results"
in our study,5 in which we discuss the relative amounts of
certain membrane proteins in the erythrocyte membrane compared with
whole microvesicles and nanovesicles (and not to vesicle membranes as
stated by the authors). There are 2 misunderstandings in the comments
of Minetti and Ciana. First, we did not "normalize AChE activity to
hemoglobin content." We mentioned the enrichment of AChE relative to
hemoglobin in microvesicles (80 times) solely to explain the low
abundance of cytosolic proteins in the vesicles (silver stain5(Fig
2)). Second, as stated in "Results," we used the raft marker
AChE for normalization to compare the relative amounts of other raft proteins, particularly stomatin and the flotillins, and contrasted these findings to nonraft proteins. The term "relative" in the criticized statement ("the relative amounts of the major
integral membrane proteins band 3 and glycophorins are diminished
[italics added]"5) should be understood as
relative to AChE and not relative to phospholipid/cell surface.
However, in contrast to the unpublished results of Minetti and Ciana
(the Butikofer et al3 reference cited by the authors does
not contain original data on the band 3 distribution), Hagelberg and
Allan reported that "band 3 and glycophorins are depleted from
microvesicles"6 using phospholipid content for
normalization. They found that only 40% of the band 3 protein is
present in the microvesicles when compared with erythrocyte membranes. To address the question of membrane protein dynamics during
microvesiculation in more detail, we compared erythrocyte membranes and
increasing amounts of microvesicles by quantitative immunoblotting, thereby assessing the protein distribution relative to AChE (data not
shown). As an example, we show the relative abundance of the respective
proteins at a 4-fold AChE activity in the microvesicle sample (Figure
1A).
Whereas similar fractions of the total erythrocyte AChE, stomatin,
flotillin-2, and aquaporin-1 content are found in lipid rafts (Figure
1B), the relative amounts of these proteins in microvesicles are quite
different, thereby demonstrating the segregation of erythrocyte raft
proteins during vesiculation. A small fraction of band 3 protein is
also found in the lipid rafts; however, the significance of this
finding remains to be evaluated. Taking the well-supported factor of 3 for the AChE enrichment in microvesicles relative to
phospholipid,4,6,7 we calculated the enrichment/depletion
of the following proteins in microvesicles relative to membrane
surface: stomatin (1.7), flotillin-2 (0.2), aquaporin-1 (0.4),
glycophorin C (0.5), and band 3 (0.4; in agreement with Hagelberg and
Allan6).
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| Figure 1.
Distribution of erythrocyte membrane proteins in
microvesicles and lipid rafts.
(A) Erythrocyte membranes (lane 1) and microvesicles (lane 2) were
prepared and AChE activity determined as described.5
Aliquots were loaded on an 11% polyacrylamide gel, with a 4-fold AChE
activity in the microvesicular sample, and analyzed by silver staining
(upper panel) and Western blotting, as indicated and previously
described5 (anti-AQP-1 was from Calbiochem, La Jolla,
CA). AChE activity is given in arbitrary units. Note the
differential distribution of stomatin and flotillin-2. (B) Erythrocyte
lipid rafts were prepared by method B1 and AChE activity
was determined. Aliquots with equal AChE activity of lipid rafts (lane
1) and erythrocyte membranes (lane 2) were loaded on an 11%
polyacrylamide gel and analyzed by silver staining (upper panel) and
Western blotting, as indicated. CA indicates carbonic anhydrase; GPC,
glycophorin C; and AQP-1, aquaporin-1.
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In accordance with our previous study,5 these data clearly
show the segregation of lipid raft proteins during calcium-induced vesiculation of erythrocytes. Whereas AChE and stomatin are enriched in
the released microvesicles, the flotillins and aquaporin-1 are
depleted. In line with the interpretation of Hagelberg and Allan,6 we assume a partial cytoskeletal association as
the cause for the depletion of the respective membrane proteins in microvesicles. The mechanism of the enrichment of AChE and stomatin in
microvesicles remains to be elucidated; however, the membrane aggregating and fusogenic properties of synexin, which is present in
vesicular rafts,5 may play an important role in this process.
Ulrich Salzer and Rainer Prohaska
Correspondence: Rainer Prohaska, University of Vienna,
Vienna Biocenter, Institute of Medical Biochemistry, Dr. Bohr-Gasse
9/3, Vienna A-1030, Austria; e-mail:
prohaska{at}bch.univie.ac.at
Acknowledgments
Supported by grant P15486 from the Austrian Science Fund.
References
1.
Salzer U, Prohaska R.
Stomatin, flotillin-1, and flotillin-2 are major integral proteins of erythrocyte lipid rafts.
Blood.
2001;97:1141-1143[Abstract/Free Full Text].
2.
Lauer S, VanWye J, Harrison T, et al.
Vacuolar uptake of host components, and a role for cholesterol and sphingomyelin in malarial infection.
EMBO J.
2000;19:3556-3564[CrossRef][Medline]
[Order article via Infotrieve].
3.
Samuel BU, Mohandas N, Harrison T, et al.
The role of cholesterol and glycosylphosphatidylinositol-anchored proteins of erythrocyte rafts in regulating raft protein content and malarial infection.
J Biol Chem.
2001;276:29319-29329[Abstract/Free Full Text].
4.
Butikofer P, Kuypers FA, Xu CM, Chiu DT, Lubin B.
Enrichment of two glycosyl-phosphatidylinositol-anchored proteins, acetylcholinesterase and decay accelerating factor, in vesicles released from human red blood cells.
Blood.
1989;74:1481-1485[Abstract/Free Full Text].
5.
Salzer U, Hinterdorfer P, Hunger U, Borken C, Prohaska R.
Ca(++)-dependent vesicle release from erythrocytes involves stomatin-specific lipid rafts, synexin (annexin VII), and sorcin.
Blood.
2002;99:2569-2577[Abstract/Free Full Text].
6.
Hagelberg C, Allan D.
Restricted diffusion of integral membrane proteins and polyphosphoinositides leads to their depletion in microvesicles released from human erythrocytes.
Biochem J.
1990;271:831-834[Medline]
[Order article via Infotrieve].
7.
Allan D, Thomas P, Limbrick AR.
The isolation and characterization of 60 nm vesicles (`nanovesicles') produced during ionophore A23187-induced budding of human erythrocytes.
Biochem J.
1980;188:881-887[Medline]
[Order article via Infotrieve].
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