Vol. 188, No. 2, 1992 October
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
Pages
30, 1992
GLYCOSYLATION
OF ANNEXIN
I AND ANNEXIN
m-558
II
Francine Goulet, K. Gregory Moore and Alan C. Sartorelli* Department of Pharmacology and Developmental Therapeutics Program, Comprehensive Cancer Center, Yale University School of Medicine, New Haven, CT 065 10
Received
August
24,
1992
Summary: Human placental annexin I and annexin II were shown to be glycosylated by one-dimensional affinity blotting with the lectin concanavalin A, which recognizes Dmannose and D-glucose residues. Further evidence that annexin I and annexin II are lycosylated was provided by the finding that these proteins incorporated D-[2,64 Hlmannose and D-[6-3Hlglucose when they were biosynthesized by the human squamous carcinoma cell line SqCC/Yl. Annexin I and annexin II could be rapidly purified from a human placental membrane extract by concanavalin A-Sepharose, which indicated that these proteins contain two biantennary mannosyl residues. 0 1992 Academic Press,
Inc.
The annexins are a family of abundant intracellular proteins that reversibly bind to phospholipids in the plasma membrane of cells by a Ca 2+-dependent process, but whose physiological function(s) remains unclear (for review, see l-3). The annexins (also called lipocortins and calpactins) have been implicated in many processes, including inflammation, regulation of membrane traffic, and intracellular signal transduction (l-3). Annexin I and annexin II possess unique amino terminal regions that are the site of posttranslational modifications which are thought to regulate the activities of these proteins (l3). For example, epidermal growth factor receptor kinase can phosphorylate a tyrosine residue in the amino terminal region of annexin I (45) and protein kinase C can phosphorylate a serine residue in the amino terminal regions of annexin I and annexin II (6). The enzyme transglutaminase can covalently cross-link annexin I to endogenous proteins by reacting with glutamine and lysine residues in its amino terminal region (7,8). We have recently shown that covalently cross-linked forms of annexin I are incorporated
* To whom reprint requests should be addressed. Abbreviations: SDS-PAGE, sodium dodecanoyl sulfate-polyacrylamide gel electrophoresis; TBS, Tris-buffered saline (50 mM Tris-HCl, 1.50 mM NaCl, pH 7.4); PBS, phosphatebuffered saline (50 mM sodium phosphate, 150 mM NaCl, pH 7.2); RIPA, radioimmune precipitation buffer (50 mM Tris-HCl, 500 mM NaCl, 25 mM EDTA, 1% Triton X-100, 0.5% sodium deoxycholate and 0.1% SDS, pH 8.0); EGTA, ethylene glycol bis (Oaminoethyl ether) N,N’-tetraacetic acid. 0006-291X/92 $4.00 Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
554
Vol.
188,
No.
2,
1992
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
into the cornified cell envelope of the squamous carcinoma cell line SqCC/Yl during differentiation (9). In this report, we describe the post-translational glycosylation of annexin I and annexin II which may occur in the amino terminal regions of these proteins. MATERIALS
AND METHODS
One dimensional immunoblottine and concanavalin A affinitv blotting: Annexin I and annexin II were isolated from human placental membranes as previously described (lo), subjected to 5 to 15% SDS-PAGE (1 l), and transferred to nitrocellulose filter papers (12). The filter papers were blocked with 5% bovine serum albumin (Sigma Chemical Co., St. Louis, MO) diluted in TBS for 2 h. Nitrocellulose filter papers were treated with monoclonal antibodies to either annexin I, annexin II, or the pl 1 subunit of the annexin II tetramer (2 pg/blot) (Zymed Laboratories, San Francisco, CA) and [1251]-labeled rabbit anti-mouse IgG (typically 106 cpm/blot) (DuPont-New England Nuclear, Boston, MA) as previously described (9). Autoradiograms were exposed overnight at -700C using XOMAT AR film (Kodak, Rochester, NY). For concanavalin A affinity blotting, the nitrocellulose filters were incubated with 5 mg/ml of peroxidase-labeled concanavalin A (Sigma) for 2 h, washed 4 times with TBS, and concanavalin A affinity was revealed using TBS containing 0.03% H202 and 0.05% 3,3’-diaminobenzidine (Sigma). Metabolic labeling and immunonrecinitation of annexin I and annexin II: The human squamous carcinoma cell line SqCUYl was grown in 25 cm2 flasks as previously described (9) and metabolically labeled in RPM1 1640 medium (Select Amine kit) (GibcoBRL, Gaithersburg, MD) containing essential amino acids without serum. Either 10 mCi/ml of L-[35S]methionine (sp. act., 1128 Ci/mmol) (DuPont-NEN) was added to the RPM1 1640 medium, or 0.5-1.0 mCi/ml of D-[6-3Hlglucose (sp. act., 23 Ci/mmol) and D-[2,6-3Hlmannose (sp. act., 47.6 Ci/mmol) (Amersham, Arlington Heights, IL) was added to the RPM1 1640 medium containing one-tenth of the normal glucose concentration for metabolic labeling of SqCC/Yl cells. After labeling for 16 h, the culture supernatant was discarded and the cells were rinsed three times in PBS. Cells were lysed in RIPA containing phenylmethylsulfonyl fluoride and aprotinin (both from Sigma), for 30 min on ice. Samples from cell lysates were adjusted to either equal cell numbers or to 106 cpm per immunoprecipitation assay. Radiolabeled cell extracts were incubated overnight at 4oC with l-2 l,tg of the anti-annexin I and II monoclonal antibodies diluted in TBS. The immune complexes were precipitated by incubation with Protein A-Sepharose (Pharmacia LKB Biotechnology Inc., Piscataway NJ) for 2 h at 4OC. The immunoprecipitates were washed 4 times with TBS by repeated centrifugations, dissolved in sample buffer (62.5 mM Tris-HCl, 6% SDS, 10% B-mercaptoethanol, 10% glycerol, pH 6.8), boiled for 5 min, and subjected to SDS-PAGE. The gels were treated with Enlightning (DuPont-NEN) and dried. Autoradiograms were exposed at 4’C for 72 h for [35S]-labeled gels and at 700C for 3 months for [3H]-labeled gels using X-OMAT AR film (Kodak). Purification of annexin I and annexin II bv concanavalin A-Senharose: Human placental membranes were isolated as previously described (10) and calcium binding proteins were extracted by treating the membranes with TBS containing 10 mM EGTA. One ml of the EGTA extract (0.42 mg of protein) was mixed with 50 ~1 of concanavalin A-Sepharose (Pharmacia) for 2 h at 4OC. The concanavalin A-Sepharose was pelleted by a brief centrifugation (4 s, 14,000g) and washed with 50 mM Tris-HCl buffer, 1 M NaCl, pH 8.0, and with 50 mM Tris-HCl buffer, 150 mM NaCl, pH 8.0. Concanavalin A binding proteins were eluted with TBS containing 0.2 M methyl-a-D-mannopyranoside (Sigma), subjected to SDS-PAGE, and either stained with Coomassie blue or transferred to nitrocellulose filter paper for immunoblotting. RESULTS
AND
DISCUSSION
Human placental annexin I and annexin II reacted in one-dimensional affinity blots with the lectin concanavalin A, which recognizes cc-D-mannose and a-D-glucose sugars (13,14) 555
Vol.
188,
No.
2,
1992
BIOCHEMICAL
AND
BIOPHYSICAL
kDa
kDa
A
B
C
RESEARCH
A
COMMUNICATIONS
B
C
97.4
D
97.4 66.2 45
31
21.5 31
14.4
21.5
01
02
me 1;. One-dimensional immunoblotting blotting of annexin I and annexin II. Annexin
and concanavalin
A affinity
I (lanes A and B, 35 pig/lane) and annexin II (lanes C and D, 20 p@lane) were reacted in one-dimensional blots with either specific monoclonal antibodies (anti-annexin I, lane A; anti-annexin II, lane C) or peroxidase-labeled concanavalin A (lanes B and D). Concanavalin A affinity was revealed using TBS containing 0.03% H202 and 0.05% 3,3’-diaminobenzidine.
Figure Purification of annexin I and annexin II and characterization of glycosylation structures by binding to concanavalin A-Sepharose. One ml of an EGTA extract of human placental membranes containing 0.42 mg of protein was incubated with concanavalin A-Sepharose for 2 h at 4OC. Bound proteins were eluted with TBS containing 0.2 M methyl-a-D-mannopyranoside, subjected to SDS-PAGE, and either stained with Coomassie blue (lane A), or transferred to nitrocellulose filter paper for immunoblotting with monoclonal antibodies to either annexin I (lane B) or annexin II monomer and small subunit (~11) of the annexin II tetramer (lane C).
(Figure
1).
Annexin
1 and annexin II did not react in affinity
blots with the lectin
psophocarpus tetragonolobus which recognizes N-acetyl-D-galactosamine
residues (15),
demonstrating the specificity of the reaction (data not shown). Annexin I and annexin II could be rapidly purified from an EGTA extract of human placental membranes by concanavalin A-Sepharose (Figure 2), indicating that these proteins contain two cc-linked biantennary mannosyl residues (13,14). Annexin I and annexin II can then be easily separated from each other by Mono S fast protein liquid chromatography (10,16,17). One of the advantages of this procedure is that the major proteolytic fragments of annexin I and annexin II, which lack full-length amino terminal regions, fail to be bound by concanavalin A-Sepharose (data not shown). Annexin II can form a non-covalent tetramer composed of two 36 kDa annexin II monomer proteins and two 11 kDa subunit proteins (17). The small subunit (~11) of the annexin II tetramer was also precipitated by concanavalin A-Sepharose (Figure 2, lane C). Annexin I and annexin II were immunoprecipitated
from proliferative
SqCC/Yl cells after being metabolically labeled with [3%]methionine
and confluent
or a mixture of D-
[6-3Hlglucose and D-[2,6-3Hlmannose (Figure 3). After being grown to confluence, SqCC/Yl cells begin to express early markers of terminal differentiation, such as the 556
Vol.
188, No. 2, 1992
BIOCHEMICAL
35S-Met.
AND BIOPHYSICAL
3H-Mannose 3H-Glucose II
35S-Met. I II
RESEARCH COMMUNICATIONS
3 H-Mannose 3 H-Glucose I I Annexin I
ABCDEFGH
Fhre
3; Metabolic labeling of annexin I and annexin II in SqCC/Yl cells.
Proliferative (lanes A-D) and confluent (lanes labeled for 16 h with either [3%]methionine [3H]glucose (lanes C,D,G,H). Annexin I B,D,F,H) were then immunoprecipitated from antibodies and Protein A-Sepharose.
E-H) SqCC/Yl cells were metabolically (lanes A,B,E,F), or [3H]mannose and (lanes A,C,E,G) and annexin II (lanes SqCC/Yl cell extracts with monoclonal
cornified envelope precursor protein involucrin (data not shown). The SqCC/Yl cells synthesized and glycosylated annexin II and the comified envelope precursor annexin I (9) during both growth states at comparable rates (Figure 3). These findings indicate that unlike involucrin, whose expression is induced by growing SqCC/Yl cells to confluence, annexin I is a constitutive precursor of the comified cell envelope. The small subunit (~11) of the annexin II tetramer was labeled by [35S]methionine, but was not labeled by D-[2,63Hlmannose and D-[6-3Hlglucose SqCC/Yl cells (data not shown).
during both the proliferative and confluent states of
Our observations strongly suggest that annexin I and annexin II have at least one site of asparagine-linked glycosylation which may contain two a-linked biantennary mannosyl residues. The amino acid sequence of the annexin I heavy chain indicates a potential Nlinked glycosylation site at the 42nd position of its amino terminal region (18). Similar analysis
of annexin
II shows a potential
N-linked
glycosylation
site at the 61th residue
of
its amino terminal region (19). We were unable to detect a significant drop in the molecular weight of annexin I and annexin II after digestion with the enzymes endoglycosidase F and a-mannosidase, suggesting that these proteins are either not heavily glycosylated or that only a subpopulation are glycosylated (data not shown). The role that glycosylation plays in the function(s) of annexin I and annexin II is unknown. Post-translational glycosylation plays a role in the function of many proteins and receptors. In addition to contributing to phenomena such as the stabilization of protein conformation and protection against proteolytic degradation, glycosylation may enhance signal transduction (20-22). The posttranslational modification of annexin I and annexin II provides another possible opportunity to regulate the activities of these proteins. 557
Vol.
188, No. 2, 1992
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
ACKNOWLEDGMENT This research was supported in part by U.S. Public Health Service Grant CA-02817 from the National Cancer Institute.
REFERENCES 1. Geisow, M.J., and Walker, J.H. (1986) Trends Biochem. Sci. 11, 420-423. 2. Smith, V.L., andDedman, J.R. (1986) J. Biol. Chem. 261, 1581515818. 3. Wallner, B.P. (1989) In Genes and Signal Transduction in Multistage Carcinogenesis (N.H. Colbum, Ed.), pp. 309-337. Marcel Dekker, Inc., New York. 4. Haigler, H.T., Schlaepfer, D.D., and Burgess, W.H. (1987) J. Biol. Chem. 262, 69216930. Pepinsky, R.B., and Sinclair, L.K. (1986) Nature 321, 81-84. Khanna, N.C., Tokuda, M., and Waisman, D.M. (1987) Cell Calcium 8,217-228. Ando, Y., Imamura, S., Owada, M.K., Kakunaga, T., and Kannagi, R. (1989) B&hem. Biophys. Res. Commun. 163, 944-951. 8. Ando, Y., Imamura, S., Owada, M.K., and Kannagi, R. (1991) J. Biol. Chem. 266, 1101-l 108. Moore, K.G., and Sartorelli, A.C. (1992) Exp. Cell Res. 200, 186-195. 1:: Moore, K.G., Goulet, F., and Sartorelli, A.C. (1992) Protein Expression and Purification 3: l-7. 11. Laemmli, U.K. (1970) Nature 227, 680-685. 12. Towbin, H., Staehlin, T., and Gordon, J. (1979) Proc. Natl. Acad. Sci. USA 76, 43504354. 13. Ogata, S., Muramatsu, T., and Kobata, A. (1975) J. Biochem. (Tokyo) 78, 687-696. 14. Baenziger, J.U., and Fiete, D. (1979) J. Biol. Chem. 254,2400-2407. 15. Appukuttan,P.S., and Basu, D. (1981) Anal. Biochem. 113, 235-255. 16. Glenney, J.R. Jr., Tack, B., and Powell, M.A. (1987) J. Cell Biol. 104, 503-511. 17. Powell, M.A., and Glenney, J.R. (1987) Biochem. J. 247, 321-328. 18. 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., and Pepinsky, R.B. (1986) Nature 320,77-81. 19. Kristensen, T.K., Saris, C.J.M., Hunter, T., Hicks, L.J., Noonan, D.J., Glenney, J.R. Jr. and Tack, B.F. (1986) Biochemistry 25,4497-4503. 20. Powell, L.M., and Pain, R.H. (1992) J. Mol. Biol. 224, 241-252. 21. Selvaraj, P., Carp’en, O., Hibbs, M.L., and Springer, T.A. (1989) J. Immunol. 143, 3283-3288. 22. Chen, J.Z., Stall, A.M., Herzenberg, L.A., and Herzenberg, L. A. (1990) EMBG J. 9, 2117-2124.
558
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
Pages
30, 1992
GLYCOSYLATION
OF ANNEXIN
I AND ANNEXIN
m-558
II
Francine Goulet, K. Gregory Moore and Alan C. Sartorelli* Department of Pharmacology and Developmental Therapeutics Program, Comprehensive Cancer Center, Yale University School of Medicine, New Haven, CT 065 10
Received
August
24,
1992
Summary: Human placental annexin I and annexin II were shown to be glycosylated by one-dimensional affinity blotting with the lectin concanavalin A, which recognizes Dmannose and D-glucose residues. Further evidence that annexin I and annexin II are lycosylated was provided by the finding that these proteins incorporated D-[2,64 Hlmannose and D-[6-3Hlglucose when they were biosynthesized by the human squamous carcinoma cell line SqCC/Yl. Annexin I and annexin II could be rapidly purified from a human placental membrane extract by concanavalin A-Sepharose, which indicated that these proteins contain two biantennary mannosyl residues. 0 1992 Academic Press,
Inc.
The annexins are a family of abundant intracellular proteins that reversibly bind to phospholipids in the plasma membrane of cells by a Ca 2+-dependent process, but whose physiological function(s) remains unclear (for review, see l-3). The annexins (also called lipocortins and calpactins) have been implicated in many processes, including inflammation, regulation of membrane traffic, and intracellular signal transduction (l-3). Annexin I and annexin II possess unique amino terminal regions that are the site of posttranslational modifications which are thought to regulate the activities of these proteins (l3). For example, epidermal growth factor receptor kinase can phosphorylate a tyrosine residue in the amino terminal region of annexin I (45) and protein kinase C can phosphorylate a serine residue in the amino terminal regions of annexin I and annexin II (6). The enzyme transglutaminase can covalently cross-link annexin I to endogenous proteins by reacting with glutamine and lysine residues in its amino terminal region (7,8). We have recently shown that covalently cross-linked forms of annexin I are incorporated
* To whom reprint requests should be addressed. Abbreviations: SDS-PAGE, sodium dodecanoyl sulfate-polyacrylamide gel electrophoresis; TBS, Tris-buffered saline (50 mM Tris-HCl, 1.50 mM NaCl, pH 7.4); PBS, phosphatebuffered saline (50 mM sodium phosphate, 150 mM NaCl, pH 7.2); RIPA, radioimmune precipitation buffer (50 mM Tris-HCl, 500 mM NaCl, 25 mM EDTA, 1% Triton X-100, 0.5% sodium deoxycholate and 0.1% SDS, pH 8.0); EGTA, ethylene glycol bis (Oaminoethyl ether) N,N’-tetraacetic acid. 0006-291X/92 $4.00 Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
554
Vol.
188,
No.
2,
1992
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
into the cornified cell envelope of the squamous carcinoma cell line SqCC/Yl during differentiation (9). In this report, we describe the post-translational glycosylation of annexin I and annexin II which may occur in the amino terminal regions of these proteins. MATERIALS
AND METHODS
One dimensional immunoblottine and concanavalin A affinitv blotting: Annexin I and annexin II were isolated from human placental membranes as previously described (lo), subjected to 5 to 15% SDS-PAGE (1 l), and transferred to nitrocellulose filter papers (12). The filter papers were blocked with 5% bovine serum albumin (Sigma Chemical Co., St. Louis, MO) diluted in TBS for 2 h. Nitrocellulose filter papers were treated with monoclonal antibodies to either annexin I, annexin II, or the pl 1 subunit of the annexin II tetramer (2 pg/blot) (Zymed Laboratories, San Francisco, CA) and [1251]-labeled rabbit anti-mouse IgG (typically 106 cpm/blot) (DuPont-New England Nuclear, Boston, MA) as previously described (9). Autoradiograms were exposed overnight at -700C using XOMAT AR film (Kodak, Rochester, NY). For concanavalin A affinity blotting, the nitrocellulose filters were incubated with 5 mg/ml of peroxidase-labeled concanavalin A (Sigma) for 2 h, washed 4 times with TBS, and concanavalin A affinity was revealed using TBS containing 0.03% H202 and 0.05% 3,3’-diaminobenzidine (Sigma). Metabolic labeling and immunonrecinitation of annexin I and annexin II: The human squamous carcinoma cell line SqCUYl was grown in 25 cm2 flasks as previously described (9) and metabolically labeled in RPM1 1640 medium (Select Amine kit) (GibcoBRL, Gaithersburg, MD) containing essential amino acids without serum. Either 10 mCi/ml of L-[35S]methionine (sp. act., 1128 Ci/mmol) (DuPont-NEN) was added to the RPM1 1640 medium, or 0.5-1.0 mCi/ml of D-[6-3Hlglucose (sp. act., 23 Ci/mmol) and D-[2,6-3Hlmannose (sp. act., 47.6 Ci/mmol) (Amersham, Arlington Heights, IL) was added to the RPM1 1640 medium containing one-tenth of the normal glucose concentration for metabolic labeling of SqCC/Yl cells. After labeling for 16 h, the culture supernatant was discarded and the cells were rinsed three times in PBS. Cells were lysed in RIPA containing phenylmethylsulfonyl fluoride and aprotinin (both from Sigma), for 30 min on ice. Samples from cell lysates were adjusted to either equal cell numbers or to 106 cpm per immunoprecipitation assay. Radiolabeled cell extracts were incubated overnight at 4oC with l-2 l,tg of the anti-annexin I and II monoclonal antibodies diluted in TBS. The immune complexes were precipitated by incubation with Protein A-Sepharose (Pharmacia LKB Biotechnology Inc., Piscataway NJ) for 2 h at 4OC. The immunoprecipitates were washed 4 times with TBS by repeated centrifugations, dissolved in sample buffer (62.5 mM Tris-HCl, 6% SDS, 10% B-mercaptoethanol, 10% glycerol, pH 6.8), boiled for 5 min, and subjected to SDS-PAGE. The gels were treated with Enlightning (DuPont-NEN) and dried. Autoradiograms were exposed at 4’C for 72 h for [35S]-labeled gels and at 700C for 3 months for [3H]-labeled gels using X-OMAT AR film (Kodak). Purification of annexin I and annexin II bv concanavalin A-Senharose: Human placental membranes were isolated as previously described (10) and calcium binding proteins were extracted by treating the membranes with TBS containing 10 mM EGTA. One ml of the EGTA extract (0.42 mg of protein) was mixed with 50 ~1 of concanavalin A-Sepharose (Pharmacia) for 2 h at 4OC. The concanavalin A-Sepharose was pelleted by a brief centrifugation (4 s, 14,000g) and washed with 50 mM Tris-HCl buffer, 1 M NaCl, pH 8.0, and with 50 mM Tris-HCl buffer, 150 mM NaCl, pH 8.0. Concanavalin A binding proteins were eluted with TBS containing 0.2 M methyl-a-D-mannopyranoside (Sigma), subjected to SDS-PAGE, and either stained with Coomassie blue or transferred to nitrocellulose filter paper for immunoblotting. RESULTS
AND
DISCUSSION
Human placental annexin I and annexin II reacted in one-dimensional affinity blots with the lectin concanavalin A, which recognizes cc-D-mannose and a-D-glucose sugars (13,14) 555
Vol.
188,
No.
2,
1992
BIOCHEMICAL
AND
BIOPHYSICAL
kDa
kDa
A
B
C
RESEARCH
A
COMMUNICATIONS
B
C
97.4
D
97.4 66.2 45
31
21.5 31
14.4
21.5
01
02
me 1;. One-dimensional immunoblotting blotting of annexin I and annexin II. Annexin
and concanavalin
A affinity
I (lanes A and B, 35 pig/lane) and annexin II (lanes C and D, 20 p@lane) were reacted in one-dimensional blots with either specific monoclonal antibodies (anti-annexin I, lane A; anti-annexin II, lane C) or peroxidase-labeled concanavalin A (lanes B and D). Concanavalin A affinity was revealed using TBS containing 0.03% H202 and 0.05% 3,3’-diaminobenzidine.
Figure Purification of annexin I and annexin II and characterization of glycosylation structures by binding to concanavalin A-Sepharose. One ml of an EGTA extract of human placental membranes containing 0.42 mg of protein was incubated with concanavalin A-Sepharose for 2 h at 4OC. Bound proteins were eluted with TBS containing 0.2 M methyl-a-D-mannopyranoside, subjected to SDS-PAGE, and either stained with Coomassie blue (lane A), or transferred to nitrocellulose filter paper for immunoblotting with monoclonal antibodies to either annexin I (lane B) or annexin II monomer and small subunit (~11) of the annexin II tetramer (lane C).
(Figure
1).
Annexin
1 and annexin II did not react in affinity
blots with the lectin
psophocarpus tetragonolobus which recognizes N-acetyl-D-galactosamine
residues (15),
demonstrating the specificity of the reaction (data not shown). Annexin I and annexin II could be rapidly purified from an EGTA extract of human placental membranes by concanavalin A-Sepharose (Figure 2), indicating that these proteins contain two cc-linked biantennary mannosyl residues (13,14). Annexin I and annexin II can then be easily separated from each other by Mono S fast protein liquid chromatography (10,16,17). One of the advantages of this procedure is that the major proteolytic fragments of annexin I and annexin II, which lack full-length amino terminal regions, fail to be bound by concanavalin A-Sepharose (data not shown). Annexin II can form a non-covalent tetramer composed of two 36 kDa annexin II monomer proteins and two 11 kDa subunit proteins (17). The small subunit (~11) of the annexin II tetramer was also precipitated by concanavalin A-Sepharose (Figure 2, lane C). Annexin I and annexin II were immunoprecipitated
from proliferative
SqCC/Yl cells after being metabolically labeled with [3%]methionine
and confluent
or a mixture of D-
[6-3Hlglucose and D-[2,6-3Hlmannose (Figure 3). After being grown to confluence, SqCC/Yl cells begin to express early markers of terminal differentiation, such as the 556
Vol.
188, No. 2, 1992
BIOCHEMICAL
35S-Met.
AND BIOPHYSICAL
3H-Mannose 3H-Glucose II
35S-Met. I II
RESEARCH COMMUNICATIONS
3 H-Mannose 3 H-Glucose I I Annexin I
ABCDEFGH
Fhre
3; Metabolic labeling of annexin I and annexin II in SqCC/Yl cells.
Proliferative (lanes A-D) and confluent (lanes labeled for 16 h with either [3%]methionine [3H]glucose (lanes C,D,G,H). Annexin I B,D,F,H) were then immunoprecipitated from antibodies and Protein A-Sepharose.
E-H) SqCC/Yl cells were metabolically (lanes A,B,E,F), or [3H]mannose and (lanes A,C,E,G) and annexin II (lanes SqCC/Yl cell extracts with monoclonal
cornified envelope precursor protein involucrin (data not shown). The SqCC/Yl cells synthesized and glycosylated annexin II and the comified envelope precursor annexin I (9) during both growth states at comparable rates (Figure 3). These findings indicate that unlike involucrin, whose expression is induced by growing SqCC/Yl cells to confluence, annexin I is a constitutive precursor of the comified cell envelope. The small subunit (~11) of the annexin II tetramer was labeled by [35S]methionine, but was not labeled by D-[2,63Hlmannose and D-[6-3Hlglucose SqCC/Yl cells (data not shown).
during both the proliferative and confluent states of
Our observations strongly suggest that annexin I and annexin II have at least one site of asparagine-linked glycosylation which may contain two a-linked biantennary mannosyl residues. The amino acid sequence of the annexin I heavy chain indicates a potential Nlinked glycosylation site at the 42nd position of its amino terminal region (18). Similar analysis
of annexin
II shows a potential
N-linked
glycosylation
site at the 61th residue
of
its amino terminal region (19). We were unable to detect a significant drop in the molecular weight of annexin I and annexin II after digestion with the enzymes endoglycosidase F and a-mannosidase, suggesting that these proteins are either not heavily glycosylated or that only a subpopulation are glycosylated (data not shown). The role that glycosylation plays in the function(s) of annexin I and annexin II is unknown. Post-translational glycosylation plays a role in the function of many proteins and receptors. In addition to contributing to phenomena such as the stabilization of protein conformation and protection against proteolytic degradation, glycosylation may enhance signal transduction (20-22). The posttranslational modification of annexin I and annexin II provides another possible opportunity to regulate the activities of these proteins. 557
Vol.
188, No. 2, 1992
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
ACKNOWLEDGMENT This research was supported in part by U.S. Public Health Service Grant CA-02817 from the National Cancer Institute.
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