Monoclonal antibodies were raised against trypsinized human skin epidermal cells and selected for their staining of the epidermal cells in a cell periphery pattern. One antibody, CP-1, immunoprecipitated a 36 kDa protein that was identified as annexin II heavy chain by microsequencing of a CNBr-generated peptide fragment from the antigen and by cross-identification with another anti-annexin II antibody. In addition to staining a broad cell periphery band in keratinocytes, CP-1 also detected annexin II outside and in between the top layer cells before cell permeabilization. Double-labeling of annexin II and F-actin revealed a distinct topographical relationship between the two, with intercellular annexin II flanked by the submembranously located actin of the juxta-positioned cells. Annexin II was isolated from cultured keratinocytes via immunoaffinity column chromatography in one step, using the same monoclonal antibody CP-1 and was found to be resolved into multiple isoforms when analyzed by two-dimensional gel electrophoresis. The predominant components of annexin II were basic, with pI of 6.5-8.5, and some of them formed disulfide-linked monomeric multimers under non-reducing conditions. Acidic annexin II isoforms with pI 5.4-5.8 were barely detectable among the total annexin II isolated but were selectively enriched in an extracellular pool created by 0.05% ethylenediaminetetraacetic acid (EDTA) dispersion of the cultured cells into single cell suspensions. Furthermore, they can be separated from the rest of annexin II by using a different elution condition. A 46 kDa protein, the identity of which is unclear, co-eluted with the acidic isoforms in the EDTA washes. These acidic isoforms, which co-eluted with the 46 kDa protein, are suspected of corresponding to the extracellular annexin II detected immunocytochemically.

REFERENCES

Ali
S. M.
,
Geisow
M. J.
,
Burgoyne
R. D.
(
1989
).
A role for calpactin in calcium-dependent exocytosis in adrenal chromaffin cells.
Nature
340
,
313
315
Barra
R. M.
,
Fenjves
E. S.
,
Taichman
L. B.
(
1994
).
Secretion of apolipoprotein E by basal cells in cultures of epidermal keratinocytes.
J. Invest. Derm
102
,
61
66
Barton
G. J.
,
Newman
R. H.
,
Freemon
P. S.
,
Crumpton
M. J.
(
1991
).
Amino acid sequence analysis of the annexin super-gene family of proteins.
Eur. J. Biochem
198
,
749
760
Burgoyne
R. D.
,
Geisow
M. J.
(
1989
).
The annexin family of calcium-binding proteins.
Cell Calcium
10
,
1
10
Cheney
R. E.
,
Willard
M. B.
(
1989
).
Characterization of the interaction between calpactin I and fodrin (non-erythroid spectrin).
J. Biol. Chem
264
,
18068
18075
Cheng
Y.-S. E.
,
Chen
L. B.
(
1981
).
Detection of phosphotyrosine-containing 34,000 dalton protein on the framework of cells transformed with Rous sarcoma virus.
Proc. Nat. Acad. Sci. USA
78
,
2388
2392
Clark
D. M.
,
Moss
S. E.
,
Wright
N. A.
,
Crumpton
M. J.
(
1991
).
Expression of annexin VI (p68, 67 kDa-calelectrin) in normal human tissues: evidence for developmental regulation in B-and T-lymphocytes.
Histochemistry
96
,
405
412
Cooper
J. A.
,
Hunter
T.
(
1983
).
Regulation of cell growth and transformation by tyrosine-specific protein kinases: the search for important cellular substrate proteins.
Curr. Top. Microbiol. Immunol
107
,
125
161
Creutz
C. E.
,
Dowling
L. G.
,
Sando
J. J.
,
Villar Palasi
C.
,
Whipple
J. H.
,
Zaks
W. J.
(
1983
).
Characterization of the chromobindins.
J. Biol. Chem
258
,
14664
14674
Creutz
C. E.
,
Zaks
W. J.
,
Hamman
H. C.
,
Crane
S.
,
Martin
W. H.
,
Gould
K. L.
,
Oddie
K. M.
,
Parsons
S. J.
(
1987
).
Identification of chromaffin granule-binding proteins.
J. Biol. Chem
262
,
1860
1868
Creutz
C. E.
(
1992
).
The annexins and exocytosis.
Science
258
,
924
931
Elias
P. M.
,
Feingold
K. R.
(
1988
).
Lipid-related barriers and gradients in the epidermis.
Ann. NY Acad. Sci
548
,
4
14
Erikson
R. L.
,
Erikson
E.
(
1980
).
Identification of a cellular protein substrate phosphorylated by the avian sarcoma virus-transforming gene product.
Cell
21
,
829
836
Fava
R. A.
,
Nanney
L. B.
,
Wilson
D.
,
King
L. E.
Jr.
(
1993
).
Annexin-1 localization in human skin: possible association with cytoskeletal elements in keratinocytes of the stratum spinosum.
J. Invest. Derm
101
,
732
737
Flower
R. J.
(
1988
).
Lipocortin and the mechanism of action of the glucocorticoids.
Br. J. Pharmacol
94
,
987
1015
Geisow
M. J.
(
1986
).
Common domain structure of Ca2+and lipid-binding proteins.
FEBS Lett
293
,
99
103
Gerke
V.
,
Weber
K.
(
1984
).
Identity of p36K phosphorylated upon Rous sarcoma virus transformation with a protein purified from brush borders; calcium-dependent binding to non-erythroid spectrin and F-actin.
EMBO J
3
,
227
233
Glenney
J. R.
Jr.
,
Glenney
P.
(
1985
).
Comparison of Ca++-regulated events in the intestinal border.
J. Cell Biol
00
,
754
763
Glenney
J. R.
Jr.
,
Tack
B. F.
(
1985
).
Amino-terminal sequence of p36 and associated p10: identification of the site of tyrosine phosphorylation and homology with S-100.
Proc. Nat. Acad. Sci. USA
82
,
7884
7888
Glenney
J. R.
Jr.
(
1986
).
Phospholipid-dependent Ca2+binding by the 36 kDa tyrosine kinase substrate (calpactin) and its 33 kDa clone.
J. Biol. Chem
261
,
7247
7252
Glenney
J. R.
Jr.
,
Boudreau
M.
,
Galyean
R.
,
Hunter
T.
,
Tack
B.
(
1986
).
Association of the S-100-related calpactin/light chain with the NH2-terminal tail of the 36 kDa heavy chain.
J. Biol. Chem
261
,
10485
10488
Goulet
F.
,
Moore
K. G.
,
Sartorelli
A. C.
(
1992
).
Glycosylation of annexin I and annexin II.
Biochem. Biophys. Res. Commun
188
,
554
558
Greenberg
M. E.
,
Edelman
G. M.
(
1983
).
The 34 kDa pp60srcsubstrate is located at the inner face of the plasma membrane.
Cell
33
,
767
779
Greenwood
M.
,
Tsang
A.
(
1991
).
Sequence and expression of annexin VII of Dictyostelium discoidium.
Biochim. Biophys. Acta
1088
,
429
432
Harrison
H. H.
,
Miller
K. L.
,
Dickinson
C.
,
Danfeldt
J.A.
) (
1992
).
Quality assurance and reproducibility of high-resolution two-dimensional electrophoresis and silver staining in polyacrylamide gels.
Amer. J. Clin. Path
97
,
97
104
Huang
K. S.
,
Wallner
B. P.
,
Mattliano
R. J.
,
Tizard
R.
,
Burne
C.
,
Frey
A.
,
Hession
C.
,
McGray
P.
,
Sinclair
L. K.
,
Chow
E. P. C.
,
Browning
J. L.
,
Ramachandran
K. L.
,
Tang
J.
,
Smart
J. E.
,
Pepinsky
R. B.
(
1986
).
Two human 35 kD inhibitors of phospholipase A2are related to substrates of pp60srcand of the epidermal growth factor receptor/kinase.
Cell
46
,
191
199
Huber
R.
,
Schneider
M.
,
Mayr
I.
,
Römisch
J.
,
Paques
E. P.
(
1990
).
The calcium bindings siets in human annexin V by crystal structure analysis at 2.0 Å resolution. Implications for membrane binding and calcium channel activity.
FEBSLett
275
,
15
21
Huber
R.
,
Berendes
R.
,
Burger
A.
,
Schneider
M.
,
Karshikov
A.
,
Luecke
H.
(
1992
).
Crystal and molecular structure of human annexin V after refinement. Implications for structure, membrane binding and ion channel formation of the annexin family of proteins.
J. Mol. Biol
223
,
683
701
Johnston
P. A.
,
Sudhof
T. C.
(
1990
).
The lipocortin/ calpactin family of calcium-binding proteins.
Biochem. Soc. Trans
18
,
1097
1098
Karshikov
A.
,
Berendes
R.
,
Burger
A.
,
Cavalie, Lux
H.D.
,
Huber
R.
(
1992
).
Annexin V membrane interaction: an electrostatic potential study.
Eur. Biophys. J
20
,
337
344
Kristensen
T.
,
Saris
C. J. M.
,
Hunter
T.
,
Hicks
L. J.
,
Noonan
D. J.
,
Glenney
J. R.
Jr.
,
Tack
B. F.
(
1986
).
Primary structure of bovine calpactin 1 heavy chain (p36), a major cellular substrate for retroviral protein-tyrosine kinases: homology with the human phospholipase A2 inhibitor lipocortin.
Biochemistry
25
,
4497
4503
Kohler
G. T.
,
Milstein
C.
(
1975
).
Continuous cultures of fused cells secreting antibody of predefined specificity.
Nature
256
,
495
497
Laemmli
U. K.
(
1970
).
Cleavage of structural proteins during the assembly of the head of bacteriophage T4.
Nature
227
,
680
685
Lehto
V. P.
,
Virtanen
I.
,
Paasivuo
R.
,
Ralston
R.
,
Litalo
K.
(
1983
).
The p36 substrate of tyrosine-specific protein kinases co-localizes with non-erythrocyte-spectrin antigen, p230, in surface lamina of cultured fibroblasts.
EMBO J
2
,
1701
1705
Ma
A. S. P.
,
Sun
T. T.
(
1986
).
Differentiation-dependent changes in the solubility of a 195-kD protein in human epidermal keratinocytes.
J. Cell Biol
103
,
41
48
Ma
A. S. P.
,
Lorincz
A. L.
(
1988
).
Immunofluorescence localization of peripheral proteins in cultured human keratinocytes.
J. Invest. Dermatol
90
,
331
335
Milstone
L. M.
,
Edelson
R. L.
(
1988
).
Endocrine, metabolic and immunologic functions of keratinocytes.
Ann. NY Acad. Sci.
548
Nigg
E. A.
,
Cooper
J. A.
,
Hunter
T.
(
1983
).
Immunofluorescent localization of a 39,000-dalton substrate of tyrosine protein kinases to the cytoplasmic surface of the plasma membrane.
J. Cell Biol
96
,
1601
1609
O'Farrell
P. A.
,
Goodman
H. M.
,
O'Farrell
P. H.
(
1977
).
Highresolution two-dimensional elecrophoresis of basic as well as acidic proteins.
Cell
12
,
1133
1142
Pepinsky
R. B.
,
Tizard
R. T.
,
Mattaliano
R. J.
,
Sinclaire
L. K.
,
Miller
G. T.
,
Browning
J. L.
,
Chow
P.
,
Burn
C.
,
Huang
K. S.
,
Pratt
D.
,
Wachter
L.
,
Hession
C.
,
Frey
A. Z.
,
Wallner
B.
(
1988
).
Five distinct calcium and phospholipid binding proteins share homology with lipocortin I.
J. Biol. Chem
263
,
10799
10811
Pfäffle
M.
,
Ruggiero
F.
,
Hofmann
H.
,
Fernández
M. P.
,
Selmin
O.
,
Yamada
Y.
,
Garrone
R.
,
von der Mark
K.
(
1988
).
Biosynthesis, a secretion and extracellular localization of anchorin CII, a collagen-binding protein of the calpactin family.
EMBO J
7
,
2335
2342
Pollard
H. B.
,
Rojas
E.
(
1988
).
Ca2+-activated synexin forms highly selective, voltage-gated Ca2+channels in phosphatidylserine bilayer membranes.
Proc. Nat. Acad. Sci. USA
85
,
2974
2978
Radke
K.
,
Carter
C.
,
Moss
P.
,
Dehazya
P.
,
Schliwa
M.
,
Martin
S.
(
1983
).
Membrane association of a 36,000-dalton substrate for tyrosine phosphorylation in chicken embryo fibroblasts transformed by avian sarcoma viruses.
J. Cell Biol
97
,
1601
1611
Rheinwald
J. G.
,
Green
H.
(
1975
).
Serial cultivation of strains of human epidermal keratinocytes: the formation of keratinizing colonies from single cells.
Cell
6
,
331
343
Saris
C. J. M.
,
Tack
B. F.
,
Kristensen
T.
,
Glenney
J. R.
Jr.
,
Hunter
T.
(
1986
).
The cDNA sequence for the protein-tyrosine kinase substrate p36 (calpactin I heavy chain) reveals a multidomain protein with internal repeats.
Cell
46
,
201
212
Soric
J.
,
Gordon
J. A.
(
1985
).
The 36-kilodalton substrate of pp60V-SRCis myristylated in a transformation-sensitive manner.
Science
230
,
563
566
Walker
J. H.
,
Obrocki
J.
,
Sudhof
T. C.
(
1983
).
Calelectrin, a calcium-dependent membrane-binding protein associated with secretory granules in Torpedo cholinergic electromotor nerve endings and rat adrenal medulla.
J. Neurochem
41
,
139
145
Wallner
B. P.
,
Mattaliano
R. J.
,
Hession
C.
,
Cate
R. L.
,
Tizard
R.
,
Sinclair
L. K.
,
Foeller
C.
,
Chow
E. P.
,
Browning
J. L.
,
Ramachandran
K. L.
,
Pepinsky
R. B.
(
1986
).
Cloning and expression of human lipocortin, a phospholipase A2 anti-inflammatory activity.
Nature
320
,
77
81
Weber
K.
,
Johnsson
N.
(
1986
).
Repeating sequence homologies in the p36 target protein of retroviral kinases and lipocortin, the p37 inhibitor of phospholipase A2.
FEBS Lett
203
,
95
98
This content is only available via PDF.