Vol. 182, No. 2, 1992 January
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
31, 1992
Pages
501-506
THREE
DIFFERENT NCA SPECIES, CGM6/CD67, NCA-95, AND NCA-90, ARE COMPRISED IN THE MAJOR 90 TO lOO-KDa BAND OF GRANULOCYTE NCA DETECTABLE UPON SDS-POLYACRYLAMIDE GEL ELECTROPHORESIS Motomu Kuroki, Yoshino Matsuo, Tetsushi Kinugasa, and Yuji Matsuoka Department of Biochemistry, School of Medicine, Fukuoka University, 7-45-l Nanakuma, Jonan-ku, Fukuoka 814-01, Japan Received
November
25,
1991
Human granulocytes express several species of nonspecific cross-reacting antigens (NCA), glycoproteins belonging to the carcinoembryonic antigen (CEA) family. Our previous studies have shown that at least two different NCA of 95 and 90 kDa are contained in the major NCA band of 90 to 100 kDa detectable upon gel electrophoresis of immunoprecipitates obtained from the cell surfaces of granulocytes with polyclonal anti-NCA. In the present study, the 90 to lOO-kDa NCA band was found to include one more species of 100 kDa. This component was reactive with an anti-CD67 antibody as well as polyclonal anti-NCA and released from the cell surface with phosphatidylinositol-specific phospholipase C, indicating that the lOO-kDa NCA species is CD67. Both antibodies revealed high binding activities with a recombinant protein of CGMG, which has been identified in a leukocyte cDNA library as an NCA gene and found to encode a glycosyl-phosphatidylinositol-anchored heterotypic cell adhesion molecule. Furthermore, the apparent molecular mass of the deglycosylated CD67 (38 kDa) corresponded with that of the CGM6 protein. These results suggest that CD67 is equivalent to the NCA species CGMG. Q 1992 Academic Press,Inc.
NCAs, a group of glycoproteins
belonging to the CEA family (reviewed
been identified in a variety of normal and cancerous hematopoietic
cells including granulocytes
epithelial cells (2-5) and also in
(6-10) and monocytes
structure of NCA-50, an NCA species mainly produced determined
by cDNA
immunoglobulin-related
cloning
(11,12),
revealing
in l), have
(6,8).
The primary
by colonic epithelial cells, was
that
NCA-50
consists
of three
domains with about 85% sequence similarity to CEA at amino
acid level. NCA-50 exhibits homotypic and heterotypic cell adhesion activities (13), as do CEA (13,14) and BGP, another
CEA family
member
(15).
On the other hand,
Abbreviations: NCA, nonspecific cross-reacting antigen; CEA, carcinoembryonic SDS-PAGE, sodium dodecyl sulfateantigen; BGP, biliary glycoprotein; polyacrylamide gel electrophoresis; GPI, glycosyl-phosphatidylinositol; CGM, CEA gene family member; PI-PLC, phosphatidylinositol-specific phospholipase C; CHO, Chinese hamster ovary. 0006-291x/92 501
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several NCA species distinct from NCA-50 have been found in granulocytes Upon SDS-PAGE of immunoprecipitates obtained from the granulocyte membrane
with polyclonal
anti-NCA,
we have detected
(7-10). surface
three NCA bands of about
160, 90 to 100, and 26 kDa (10). The most prominent band, the 90 to lOO-kDa one, was composed of at least two different GPI-anchored NCA species of about 95 (NCA95) and 90 kDa (NCA-90) (10). clones belonging
Subsequently,
we have identified nine different cDNA
to the CEA gene family including CGMla,
CGMl b, CGM6 (W272),
CGM7, NCA-50, and four isoforms of BGP in a library of human leukocytes and demonstrated
that the CGM6 clone encodes
a GPI-anchored
(16-18)
heterotypic
cell
adhesion molecule specifically interacting with NCA-50 (19). Berling et al. (20) suggested that NCA-95 is encoded by CGM6 mainly on the basis of reactivities with monoclonal
antibodies.
However, the peptide size of our NCA-95 (45 kDa) did not
coincide with that of the recombinant
protein of CGM6 (37 kDa) (16,17), and these two
proteins showed different reactivities against monoclonal antibodies for CEA (17). Recently, it was suggested that CD67, a granulocyte-specific cell surface marker with an apparent molecular mass of about 100 kDa (21,22), is a member of the CEA gene family (1,23). CGM6 is equivalent detectable 90 kDa.
In the present study, we found by using an anti-CD67 antibody that to CD67 contained
in the major 90 to lOO-kDa
NCA band
with polyclonal anti-NCA along with the two NCA species of about 95 and
MATERIALS
AND
METHODS
Preparation of human granulocytes. Granulocytes were isolated from peripheral blood of healthy volunteers by dextran sedimentation followed by centrifugation on the gradients of Ficoll-Paque (Pharmacia). Purity of neutrophils obtained was more than 95% as judged by morphological observations, and cell viability was greater than 98% as determined by trypan blue exclusion. Antibodies and enzyme immunoassay. Preparation and purification of a polyclonal rabbit antibody against NCA isolated from human lung were performed according to the method previously described (24). The antibody was used without absorption with CEA. Two anti-CEA monoclonal antibodies, F34-187 and F36-54, are reactive with both CEA and NCA-50 but recognize different determinants (25). An antiCD67 monoclonal antibody clone, 80H3, was obtained from lmmunotech (Marseille, France). Reactivities of granulocytes and CHO cells expressing CGM6 (17) with the antibodies were examined by the avidin-biotin-based enzyme immunoassay system previously described (25). Briefly, the cells fixed with 0.1% glutaraldehyde on 96-well culture plates were first incubated with the rabbit or mouse antibodies, then with biotinylated antibodies against rabbit or mouse IgG, and finally with avidin-conjugated peroxidase. The enzyme activity was developed by adding Hz02 and o-phenylenediamine, and absorbance at 492 nm was measured. Radiolabeling, of neutrophils
immunoprecipitation and SDS-PAGE analysis. Cell surfaces were labeled with 1251 by the lodogen method (25). In certain 502
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BIOCHEMICAL
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BIOPHYSICAL
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experiments, the labeled cells were treated with 0.5 units/ml PI-PLC (Funakoshi, Tokyo) in Ca++ and Mg++-free Dulbecco’s phosphate-buffered saline for 1 h at 37°C. Cell lysates in a buffer containing 1% Nonidet P-40 (17) were incubated with polyclonal rabbit anti-NCA, 80H3, or biotinylated F34-187 and F36-54 (25), which were then precipitated with Pansorbin (Calbiochem), protein A-conjugated agarose, or streptoavidin-conjugated agarose (Pierce), respectively (17,25). Antigens thus precipitated were analyzed by SDS-PAGE (26) using 816% gradient gels under reduced conditions. For deglycosylation, the samples were treated with N-glycanase (Genzyme) according to the supplier’s instruction. The bands were visualized by autoradiography and their relative molecular masses were estimated by comparison of their migration distances with those of [14C]methylated proteins obtained from Amersham, including myosin (200 kDa), phosphorylase b (97.4 kDa), bovine serum albumin (69 kDa), ovalbumin (46 kDa), and carbonic anhydrase (30 kDa). RESULTS
AND
DISCUSSION
Figure 1 shows the reactivities of the antibodies used in this study with granulocytes and CHO cells expressing CGMG. All antibodies revealed substantial reactivities with granulocytes upon enzyme immunoassay (Fig. 1A). As can be seen in Fig. 1 B, the antiCD67 antibody 80H3 and rabbit anti-NCA exhibited high binding activities with CGM6 expressed
on CHO cells, while F34-187
and F36-54 gave a low and a negligible
reactivity, respectively, as previously described (17). We then analyzed the molecule recognized by 80H3 on the cell surface of granulocytes by SDS-PAGE. As previously reported (lo), F34-187 identifies three NCA species, NCA-160, -95, and -26 (Fig. 2, lane 3), and F36-54 reacts with NCA-95 and -90 which appear as a single broad band (lane 4).
Here 80H3 was found to
0 II
1
A&o&es,
‘noog/rnl
Fioure 1. Reactivities of granulocytes and CHO cells expressing CGM6 with the CD67 Granulocytes (panel A) and CHO cells antibody 80H3 and anti-NCA antibodies. expressing CGM6 (panel B) were examined for reactivities with 80H3, rabbit anti-NCA, F34-187, and F36-54 by the avidin-biotin-based enzyme immunoassay as described in “Materials and Methods”. All antibodies showed negligible responses to parental CHO cells.
Vol.
182, No. 2, 1992
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AND BIOPHYSICAL
N-Glycanase
(4 2
3
4
5
6
7
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t-9 8
9
80H3 PI-PT -+-
10
Ra-NCA +
100K
02
03 Fiaure 2 Comparison of CD67 and the NCA species of 95 and 90 kDa by SDSPAGE. lmmunoprecipitates from cell lysates of IW-labeled granulocytes with polyclonal anti-NCA (lanes 1 and 5) 80H3 (lanes 2, 6, 9, and lo), F34-187 (lanes 3 and 7), and F36-54 (lanes 4 and 8), were treated with (lanes 5-8) or without Nglycanase (lanes 1-4, 9, and lo), and subjected to SDS-PAGE/autoradiography as described in “Materials and Methods”. The lysate for lane 10 was pre-absorbed with polyclonal anti-NCA before precipitation with 80H3. The apparent molecular masses of the deglycosylated antigens were indicated by the numbers (kDa) in the lanes. The two bands of about 40 and 50 kDa seen in lane 5 are also visible in lane 6, probably representing incompletely deglycosylated forms of CD67. FZ Release of CD67 and the 90-l 00 kDa NCA molecules with PI-PLC from the granulocyte cell surface. Granulocytes labeled with ts5l were incubated with (lanes 2 and 4) or without PI-PLC (lanes 1 and 3), and immunoprecipitates from the incubation medium with 80H3 (lanes 1 and 2) and polyclonal anti-NCA (lanes 3 and 4) were analyzed by SDS-PAGElautoradiography as described in “Materials and Methods”.
precipitate
a single component
This 100-kDa component,
of about 100 kDa (lane 2) larger than NCA-95 (lane 3).
corresponding
in the 90 to lOO-kDa band obtained confirmed reduced
to CD67 (21,22), appeared
with polyclonal
anti-NCA
to be contained
(lane 1).
This was
by the following results: 1) The intensity of the CD67 band was significantly by pre-absorption
of the lysate with polyclonal
anti-NCA
(Fig. 2, lane lo),
indicating that the lOO-kDa CD67 is reactive with both polyclonal anti-NCA 2) The deglycosylated
and 80H3.
form of CD67 was found to be about 38 kDa (Fig. 2, lane 6), and
this band could be seen in the precipitates with the deglycosylated
with the polyclonal antibody (lane 5) along
forms of NCA-160 (60 kDa) (lane 7), NCA-95 (45 kDa) (lanes
7 and 8), and NCA-90 (28 kDa) (lane 8). 3) The 90 to lOO-kDa molecules reactive with polyclonal anti-NCA
(Fig. 3, lane 4) as well as CD67 (lane 2) were released from the
cell surface with PI-PLC.
Thus it was shown that two NCA species of 95 and 90 kDa
and CD67, all GPI-anchored
membrane
proteins (10,27), constitute
the major NCA
band of around 90 to 100 kDa detectable with polyclonal anti-NCA. Although the cDNA clones for CGM6 were obtained from libraries of leukocytes
(16)
and the spleen of a chronic myeloid leukemia patient (20) we have not been able to
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BIOCHEMICAL
identify its corresponding of the deglycosylated
AND
BIOPHYSICAL
protein in granulocytes
RESEARCH
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(17). The apparent
molecular mass
CD67 (38 kDa) (Fig. 2) was similar to that of the unglycosylated
peptide of CGM6 expressed on COS-1 cells treated with tunicamycin (37 kDa) (16) and also to the molecular mass of the CGM6 peptide calculated from its amino acid sequence (32 kDa plus mass of the GPI moiety) (16). Furthermore, and ref. 27) and CGM6 (16,20) are GPI-anchored
membrane
both CD67 (Fig. 3
proteins.
These findings,
together with the result that CGM6 was reactive with the anti-CD67 antibody (Fig. 1 B), strongly suggest that CD67 is equivalent to CGMG. In spite of its low but significant binding to CGM6 shown by enzyme immunoassay (Fig. lB), F34-187 failed to precipitate the lOO-kDa CD67 on granulocytes.
This is
probably because the affinity of F34-187 to CGM6 is too low to precipitate a detectable amount of the antigen under the conditions used, and explains why we have previously failed to identify CGM6 at protein level in granulocytes (17). Our NCA-95 is therefore
a different species from the NCA-95 that Berling et al. (20) suggested
CGMG. Several possible functions are suggested
for the CEA family members:
to be
homotypic
and heterotypic cell adhesion activities for CEA (13,14), NCA-50 (13), and BGP (15); ectoATPase
activity for BGP (28); binding of Escherichia
co/i for CEA and NCA-50
(29). Unlike the other family members, CGM6 acts in vitro as a heterotypic,
but not a
homotypic, cell adhesion molecule specifically interacting with NCA-50 (19). Although cDNA clones corresponding to NCA-50 were found in a library of leukocytes (17) we have failed to identify NCA-50 at protein level in granulocytes (9,10), suggesting that granulocytes important
do not produce
a detectable
role as a cell adhesion
amount of NCA-50.
molecule when granulocytes
CGM6 may play an infiltrate the mucosal
membrane of the gastrointestinal tracts where NCA-50 are abundantly Alternatively, CGM6 may interact with the other NCA species expressed surface of nearby granulocytes in the interaction
between
for cell contact. granulocytes
expressed. on the cell
The possibility that CGM6 participates
and endothelial
cells should
also be
considered. ACKNOWLEDGMENT
This work was supported in part by a Grant-in-Aid for Cancer Research from Ministry of Education, Science and Culture, Japan. REFERENCES
1.
Thompson,
J. H., Grunert, F., and Zimmermann,
W. (1991) J. Clin. Lab. Anal. 5,
344-366. 2.
Von Kleist, S., Chavanel, G., and Burtin, P. (1972) 69, 2492-2494. 505
Proc. Natl. Acad. Sci. USA
Vol.
3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20.
21. 22.
23. 24. 25. 26. 27.
28. 29.
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AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
Mach, J. -P., and Pusztaszeri, G. (1972) Immunochemistry 9,1031-1034. Kessler, M. J., Shively, J. E., Pritchard, D. G., and Todd, C. W. (1978) Cancer Res. 38, 1041-l 048. Grunert, F., AbuHarfeil, N., Schwarz, K., and von Kleist, S. (1985) Int. J. Cancer 36, 357-362. Burtin, P., Quan, P. C., and Sabine, M. C. (1975) Nature 255, 714-716. Buchegger, F., Schreyer, M., Carrel, S., and Mach, J. -P. (1984) Int. J. Cancer 33, 643-649. Audette, M., Buchegger, F., Schreyer, M., and Mach, J. -P. (1987) Mol. Immunol. 24, 1177-l 186. Kuroki, MO., Kuroki, Ma., Moore, G. E., Ichiki, S., and Matsuoka, Y. (1988) Jpn. J. Cancer Res. (Gann) 79,82-90. Kuroki, MO., Matsuo, Y., Kuroki, Ma., and Matsuoka, Y. (1990) Biochem. Biophys. Res. Commun. 166,701-708. Tawaragi, Y., Oikawa, S., Matsuoka, Y., Kosaki, G., and Nakazato, H. (1988) Biochem. Biophys. Res. Commun. 150,89-96. Neumaier, M., Zimmermann, W., Shively, L., Hinoda, Y., Riggs, A. D., and Shively, J. E. (1988) J. Biol. Chem. 263, 3202-3207. Oikawa, S., Inuzuka, C., Kuroki, MO., Matsuoka, Y., Kosaki, G., and Nakazato, H. (1989) Biochem. Biophys. Res. Commun. 164, 39-45. Benchimol, S., Fuks, A., Jothy, S., Beauchemin, N., Shirota, K., and Stanners, C. P. (1989). Cell 57, 327-334. Rojas, M., Fuks, A., and Stanners, C. P. (1990) Cell Growth Differ. 1, 527-533. Arakawa, F., Kuroki, MO., Misumi, Y., Oikawa, S., Nakazato, H., and Matsuoka, Y. (1990) Biochem. Biophys. Res. Commun. 166, 1063-1071. Kuroki, MO., Arakawa, F., Matsuo, Y., Oikawa, S., Misumi, Y., Nakazato, H., and Matsuoka, Y. (1991) J. Biol. Chem. 266, 11810-l 1817. Kuroki, MO., Arakawa, F., Matsuo, Y., Oikawa, S., Nakazato, H., and Matsuoka, Y. (1991) Biochem. Biophys. Res. Commun. 176, 578-585. Oikawa, S., Inuzuka, C., Kuroki, MO., Arakawa, F., Matsuoka, Y., Kosaki, G., and Nakazato, H. (1991) J. Biol. Chem. 266, 7995-8001. Berling, B., Kolbinger, F., Grunert, F., Thompson, J. A., Brombacher, F., Buchegger, F., von Kleist, S., and Zimmermann, W. (1990) Cancer Res. 50, 6534-6539. Tetteroo, P. A. T., Bos, M. J. E., Visser, F. J., and von dem Borne, A. E. G. Kr. (1986) J. Imuunol. 136, 3427-3432. Stockinger, H. (1989) in Leukocyte Typing IV (W. Knapp, B. D&ken, W. R. Gilks, E. P. Rieber, R. E. Schmidt, H. Stein, and A. E. G. Kr. von dem Borne, Eds.), pp. 840-841. Oxford University, Oxford, UK. Van der Schoot, C. E., Kuijpers, T. W., Daams, M., and von dem Borne, A. E. G. Kr. (1990) Blood 76, 196 (abstr). Kuroki, Ma., Ichiki, S., Kuroki, MO., and Matsuoka, Y. (1982) J. Natl. Cancer Inst. 69, 401-408. Kuroki, MO., Matsuo, Y., Ohtani, T., Minowada, J., Kuroki, Ma., and Matsuoka, Y. (1990) Mol. Immunol. 27, 689-696. Laemmli, U. K. (1970) Nature 227, 680-685. Van der Shoot, C. E., Huizinga, T. W. J., Gadd, S., Majdic, O., Wijmans, R, Knapp, W., and von dem Borne, A. E. G. Kr. (1989) in Leukocyte Typing IV (W. Knapp, B. D&ken, W. R. Gilks, E. P. Rieber, R. E. Schmidt, H. Stein, and A. E. G. Kr. von dem Borne, Eds.), pp. 887-891. Oxford University, Oxford, UK. Lin, S. -H., and Guidotti, G. (1989) J. Biol. Chem. 264, 14408-14414. Leusch, H.-G., Hefta, S. A., Drzeniek, Z., Hummel, K., Markos-Pusztai, Z., and Wagener, C. (1990) FEBS Lett. 261,405-409.
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
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Pages
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THREE
DIFFERENT NCA SPECIES, CGM6/CD67, NCA-95, AND NCA-90, ARE COMPRISED IN THE MAJOR 90 TO lOO-KDa BAND OF GRANULOCYTE NCA DETECTABLE UPON SDS-POLYACRYLAMIDE GEL ELECTROPHORESIS Motomu Kuroki, Yoshino Matsuo, Tetsushi Kinugasa, and Yuji Matsuoka Department of Biochemistry, School of Medicine, Fukuoka University, 7-45-l Nanakuma, Jonan-ku, Fukuoka 814-01, Japan Received
November
25,
1991
Human granulocytes express several species of nonspecific cross-reacting antigens (NCA), glycoproteins belonging to the carcinoembryonic antigen (CEA) family. Our previous studies have shown that at least two different NCA of 95 and 90 kDa are contained in the major NCA band of 90 to 100 kDa detectable upon gel electrophoresis of immunoprecipitates obtained from the cell surfaces of granulocytes with polyclonal anti-NCA. In the present study, the 90 to lOO-kDa NCA band was found to include one more species of 100 kDa. This component was reactive with an anti-CD67 antibody as well as polyclonal anti-NCA and released from the cell surface with phosphatidylinositol-specific phospholipase C, indicating that the lOO-kDa NCA species is CD67. Both antibodies revealed high binding activities with a recombinant protein of CGMG, which has been identified in a leukocyte cDNA library as an NCA gene and found to encode a glycosyl-phosphatidylinositol-anchored heterotypic cell adhesion molecule. Furthermore, the apparent molecular mass of the deglycosylated CD67 (38 kDa) corresponded with that of the CGM6 protein. These results suggest that CD67 is equivalent to the NCA species CGMG. Q 1992 Academic Press,Inc.
NCAs, a group of glycoproteins
belonging to the CEA family (reviewed
been identified in a variety of normal and cancerous hematopoietic
cells including granulocytes
epithelial cells (2-5) and also in
(6-10) and monocytes
structure of NCA-50, an NCA species mainly produced determined
by cDNA
immunoglobulin-related
cloning
(11,12),
revealing
in l), have
(6,8).
The primary
by colonic epithelial cells, was
that
NCA-50
consists
of three
domains with about 85% sequence similarity to CEA at amino
acid level. NCA-50 exhibits homotypic and heterotypic cell adhesion activities (13), as do CEA (13,14) and BGP, another
CEA family
member
(15).
On the other hand,
Abbreviations: NCA, nonspecific cross-reacting antigen; CEA, carcinoembryonic SDS-PAGE, sodium dodecyl sulfateantigen; BGP, biliary glycoprotein; polyacrylamide gel electrophoresis; GPI, glycosyl-phosphatidylinositol; CGM, CEA gene family member; PI-PLC, phosphatidylinositol-specific phospholipase C; CHO, Chinese hamster ovary. 0006-291x/92 501
$1.50
Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
Vol.
182, No. 2, 1992
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
several NCA species distinct from NCA-50 have been found in granulocytes Upon SDS-PAGE of immunoprecipitates obtained from the granulocyte membrane
with polyclonal
anti-NCA,
we have detected
(7-10). surface
three NCA bands of about
160, 90 to 100, and 26 kDa (10). The most prominent band, the 90 to lOO-kDa one, was composed of at least two different GPI-anchored NCA species of about 95 (NCA95) and 90 kDa (NCA-90) (10). clones belonging
Subsequently,
we have identified nine different cDNA
to the CEA gene family including CGMla,
CGMl b, CGM6 (W272),
CGM7, NCA-50, and four isoforms of BGP in a library of human leukocytes and demonstrated
that the CGM6 clone encodes
a GPI-anchored
(16-18)
heterotypic
cell
adhesion molecule specifically interacting with NCA-50 (19). Berling et al. (20) suggested that NCA-95 is encoded by CGM6 mainly on the basis of reactivities with monoclonal
antibodies.
However, the peptide size of our NCA-95 (45 kDa) did not
coincide with that of the recombinant
protein of CGM6 (37 kDa) (16,17), and these two
proteins showed different reactivities against monoclonal antibodies for CEA (17). Recently, it was suggested that CD67, a granulocyte-specific cell surface marker with an apparent molecular mass of about 100 kDa (21,22), is a member of the CEA gene family (1,23). CGM6 is equivalent detectable 90 kDa.
In the present study, we found by using an anti-CD67 antibody that to CD67 contained
in the major 90 to lOO-kDa
NCA band
with polyclonal anti-NCA along with the two NCA species of about 95 and
MATERIALS
AND
METHODS
Preparation of human granulocytes. Granulocytes were isolated from peripheral blood of healthy volunteers by dextran sedimentation followed by centrifugation on the gradients of Ficoll-Paque (Pharmacia). Purity of neutrophils obtained was more than 95% as judged by morphological observations, and cell viability was greater than 98% as determined by trypan blue exclusion. Antibodies and enzyme immunoassay. Preparation and purification of a polyclonal rabbit antibody against NCA isolated from human lung were performed according to the method previously described (24). The antibody was used without absorption with CEA. Two anti-CEA monoclonal antibodies, F34-187 and F36-54, are reactive with both CEA and NCA-50 but recognize different determinants (25). An antiCD67 monoclonal antibody clone, 80H3, was obtained from lmmunotech (Marseille, France). Reactivities of granulocytes and CHO cells expressing CGM6 (17) with the antibodies were examined by the avidin-biotin-based enzyme immunoassay system previously described (25). Briefly, the cells fixed with 0.1% glutaraldehyde on 96-well culture plates were first incubated with the rabbit or mouse antibodies, then with biotinylated antibodies against rabbit or mouse IgG, and finally with avidin-conjugated peroxidase. The enzyme activity was developed by adding Hz02 and o-phenylenediamine, and absorbance at 492 nm was measured. Radiolabeling, of neutrophils
immunoprecipitation and SDS-PAGE analysis. Cell surfaces were labeled with 1251 by the lodogen method (25). In certain 502
Vol.
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No.
2, 1992
BIOCHEMICAL
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BIOPHYSICAL
RESEARCH
COMMUNICATIONS
experiments, the labeled cells were treated with 0.5 units/ml PI-PLC (Funakoshi, Tokyo) in Ca++ and Mg++-free Dulbecco’s phosphate-buffered saline for 1 h at 37°C. Cell lysates in a buffer containing 1% Nonidet P-40 (17) were incubated with polyclonal rabbit anti-NCA, 80H3, or biotinylated F34-187 and F36-54 (25), which were then precipitated with Pansorbin (Calbiochem), protein A-conjugated agarose, or streptoavidin-conjugated agarose (Pierce), respectively (17,25). Antigens thus precipitated were analyzed by SDS-PAGE (26) using 816% gradient gels under reduced conditions. For deglycosylation, the samples were treated with N-glycanase (Genzyme) according to the supplier’s instruction. The bands were visualized by autoradiography and their relative molecular masses were estimated by comparison of their migration distances with those of [14C]methylated proteins obtained from Amersham, including myosin (200 kDa), phosphorylase b (97.4 kDa), bovine serum albumin (69 kDa), ovalbumin (46 kDa), and carbonic anhydrase (30 kDa). RESULTS
AND
DISCUSSION
Figure 1 shows the reactivities of the antibodies used in this study with granulocytes and CHO cells expressing CGMG. All antibodies revealed substantial reactivities with granulocytes upon enzyme immunoassay (Fig. 1A). As can be seen in Fig. 1 B, the antiCD67 antibody 80H3 and rabbit anti-NCA exhibited high binding activities with CGM6 expressed
on CHO cells, while F34-187
and F36-54 gave a low and a negligible
reactivity, respectively, as previously described (17). We then analyzed the molecule recognized by 80H3 on the cell surface of granulocytes by SDS-PAGE. As previously reported (lo), F34-187 identifies three NCA species, NCA-160, -95, and -26 (Fig. 2, lane 3), and F36-54 reacts with NCA-95 and -90 which appear as a single broad band (lane 4).
Here 80H3 was found to
0 II
1
A&o&es,
‘noog/rnl
Fioure 1. Reactivities of granulocytes and CHO cells expressing CGM6 with the CD67 Granulocytes (panel A) and CHO cells antibody 80H3 and anti-NCA antibodies. expressing CGM6 (panel B) were examined for reactivities with 80H3, rabbit anti-NCA, F34-187, and F36-54 by the avidin-biotin-based enzyme immunoassay as described in “Materials and Methods”. All antibodies showed negligible responses to parental CHO cells.
Vol.
182, No. 2, 1992
BIOCHEMICAL
AND BIOPHYSICAL
N-Glycanase
(4 2
3
4
5
6
7
RESEARCH COMMUNICATIONS
t-9 8
9
80H3 PI-PT -+-
10
Ra-NCA +
100K
02
03 Fiaure 2 Comparison of CD67 and the NCA species of 95 and 90 kDa by SDSPAGE. lmmunoprecipitates from cell lysates of IW-labeled granulocytes with polyclonal anti-NCA (lanes 1 and 5) 80H3 (lanes 2, 6, 9, and lo), F34-187 (lanes 3 and 7), and F36-54 (lanes 4 and 8), were treated with (lanes 5-8) or without Nglycanase (lanes 1-4, 9, and lo), and subjected to SDS-PAGE/autoradiography as described in “Materials and Methods”. The lysate for lane 10 was pre-absorbed with polyclonal anti-NCA before precipitation with 80H3. The apparent molecular masses of the deglycosylated antigens were indicated by the numbers (kDa) in the lanes. The two bands of about 40 and 50 kDa seen in lane 5 are also visible in lane 6, probably representing incompletely deglycosylated forms of CD67. FZ Release of CD67 and the 90-l 00 kDa NCA molecules with PI-PLC from the granulocyte cell surface. Granulocytes labeled with ts5l were incubated with (lanes 2 and 4) or without PI-PLC (lanes 1 and 3), and immunoprecipitates from the incubation medium with 80H3 (lanes 1 and 2) and polyclonal anti-NCA (lanes 3 and 4) were analyzed by SDS-PAGElautoradiography as described in “Materials and Methods”.
precipitate
a single component
This 100-kDa component,
of about 100 kDa (lane 2) larger than NCA-95 (lane 3).
corresponding
in the 90 to lOO-kDa band obtained confirmed reduced
to CD67 (21,22), appeared
with polyclonal
anti-NCA
to be contained
(lane 1).
This was
by the following results: 1) The intensity of the CD67 band was significantly by pre-absorption
of the lysate with polyclonal
anti-NCA
(Fig. 2, lane lo),
indicating that the lOO-kDa CD67 is reactive with both polyclonal anti-NCA 2) The deglycosylated
and 80H3.
form of CD67 was found to be about 38 kDa (Fig. 2, lane 6), and
this band could be seen in the precipitates with the deglycosylated
with the polyclonal antibody (lane 5) along
forms of NCA-160 (60 kDa) (lane 7), NCA-95 (45 kDa) (lanes
7 and 8), and NCA-90 (28 kDa) (lane 8). 3) The 90 to lOO-kDa molecules reactive with polyclonal anti-NCA
(Fig. 3, lane 4) as well as CD67 (lane 2) were released from the
cell surface with PI-PLC.
Thus it was shown that two NCA species of 95 and 90 kDa
and CD67, all GPI-anchored
membrane
proteins (10,27), constitute
the major NCA
band of around 90 to 100 kDa detectable with polyclonal anti-NCA. Although the cDNA clones for CGM6 were obtained from libraries of leukocytes
(16)
and the spleen of a chronic myeloid leukemia patient (20) we have not been able to
Vol.
182,
No.
2, 1992
BIOCHEMICAL
identify its corresponding of the deglycosylated
AND
BIOPHYSICAL
protein in granulocytes
RESEARCH
COMMUNICATIONS
(17). The apparent
molecular mass
CD67 (38 kDa) (Fig. 2) was similar to that of the unglycosylated
peptide of CGM6 expressed on COS-1 cells treated with tunicamycin (37 kDa) (16) and also to the molecular mass of the CGM6 peptide calculated from its amino acid sequence (32 kDa plus mass of the GPI moiety) (16). Furthermore, and ref. 27) and CGM6 (16,20) are GPI-anchored
membrane
both CD67 (Fig. 3
proteins.
These findings,
together with the result that CGM6 was reactive with the anti-CD67 antibody (Fig. 1 B), strongly suggest that CD67 is equivalent to CGMG. In spite of its low but significant binding to CGM6 shown by enzyme immunoassay (Fig. lB), F34-187 failed to precipitate the lOO-kDa CD67 on granulocytes.
This is
probably because the affinity of F34-187 to CGM6 is too low to precipitate a detectable amount of the antigen under the conditions used, and explains why we have previously failed to identify CGM6 at protein level in granulocytes (17). Our NCA-95 is therefore
a different species from the NCA-95 that Berling et al. (20) suggested
CGMG. Several possible functions are suggested
for the CEA family members:
to be
homotypic
and heterotypic cell adhesion activities for CEA (13,14), NCA-50 (13), and BGP (15); ectoATPase
activity for BGP (28); binding of Escherichia
co/i for CEA and NCA-50
(29). Unlike the other family members, CGM6 acts in vitro as a heterotypic,
but not a
homotypic, cell adhesion molecule specifically interacting with NCA-50 (19). Although cDNA clones corresponding to NCA-50 were found in a library of leukocytes (17) we have failed to identify NCA-50 at protein level in granulocytes (9,10), suggesting that granulocytes important
do not produce
a detectable
role as a cell adhesion
amount of NCA-50.
molecule when granulocytes
CGM6 may play an infiltrate the mucosal
membrane of the gastrointestinal tracts where NCA-50 are abundantly Alternatively, CGM6 may interact with the other NCA species expressed surface of nearby granulocytes in the interaction
between
for cell contact. granulocytes
expressed. on the cell
The possibility that CGM6 participates
and endothelial
cells should
also be
considered. ACKNOWLEDGMENT
This work was supported in part by a Grant-in-Aid for Cancer Research from Ministry of Education, Science and Culture, Japan. REFERENCES
1.
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