©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Expression of the Leukocyte-associated Sialoglycoprotein CD43 by a Colon Carcinoma Cell Line (*)

Dan Baeckström (1), Ke Zhang (1), Noomi Asker (1), Ulla Rüetschi (2), Malin Ek (1), Gunnar C. Hansson (1)

From the (1) Department of Medical Biochemistry, University of Göteborg, Medicinaregatan 9A, S-413 90 Gothenburg and the (2) Department of Clinical Chemistry, University of Göteborg, Sahlgrenska Hospital, S-413 45 Gothenburg, Sweden

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The colon adenocarcinoma cell line COLO 205 secretes L-CanAg, a mucin-like glycoprotein carrying the carcinoma-associated sialyl-Lewis a carbohydrate epitope. In an attempt to identify its apoprotein, an NH-terminal peptide sequence was obtained from purified L-CanAg. In all interpretable positions, this sequence showed 100% identity to the NH-terminal of human CD43 (leukosialin, sialophorin), a plasma membrane-bound sialoglycoprotein hitherto only identified in leukocytes and other hematopoietic cells. An antiserum against deglycosylated L-CanAg and an anti-CD43 antiserum both immunoprecipitated a 61-kDa band, interpreted as the CD43 precursor, from COLO 205 cells as well as from the known CD43-expressing cell line HL-60. Results from immunoprecipitations following pulse-chase experiments and tunicamycin treatments were in agreement with earlier studies on the CD43 precursor. RNA blot analysis confirmed the expression of CD43 by the COLO 205 cell line, whereas three other colon carcinoma cell lines were negative. The glycosylation-dependent monoclonal antibody Leu-22, which recognizes leukocyte CD43, failed to bind L-CanAg, probably due to its much more extensive glycosylation. We conclude that L-CanAg is the secreted extracellular domain of a novel glycoform of CD43 and that CD43, if expressed in other carcinoma cells, may have escaped notice in studies relying on glycosylation-dependent monoclonal antibodies against leukocyte CD43.


INTRODUCTION

Mucins are a heterogeneous group of secreted or membrane-bound glycoproteins characterized by a high carbohydrate content (>50% by weight) caused by the attachment of O-glycans to serine or threonine residues in more or less repetitive amino acid sequences (1) . Mucins were first isolated as secreted oligo- or polymerized molecules from the gel-like mucus layer covering epithelial surfaces (2) . Later, however, the term mucin has also been used to describe integral membrane glycoproteins present on the apical surface of various epithelial cells (3) and even on leukocytes and other non-epithelial cells (4) . Increased abundance and/or abnormal glycosylation of mucins are often observed in carcinomas (5) .

We have previously described the isolation of two mucins carrying the carcinoma-associated sialyl-Lewis a (Si-Le; NeuAc23Gal13(Fuc14) GlcNAc1)() carbohydrate epitope from xenografts and spent culture medium of the colon adenocarcinoma cell line COLO 205 (6) . The larger (600-800 kDa) mucin, named H-CanAg, was predominantly a membrane-bound molecule and was shown to have the MUC1 gene product as apoprotein. MUC1 mucins are often abnormally expressed in carcinomas. The smaller (150-300 kDa) mucin, named L-CanAg, appeared to be a secreted molecule, and its apoprotein could not be identified.

In this paper we demonstrate that the apoprotein of L-CanAg is identical to that of CD43 (leukosialin, sialophorin) or, more likely, to an NH-terminal part of this molecule. CD43, which was originally isolated from leukocytes (7, 8) , is an integral membrane sialoglycoprotein with a mucin-like extracellular domain (9) . Its expression has until now been described as restricted to the hematopoietic cell lineage (10) .


MATERIALS AND METHODS

Cell Culture

The COLO 205 colon adenocarcinoma cell line (ATCC CCL 222) was cultured in Iscove's medium (Life Technologies, Inc., Paisley, Scotland) containing 10% fetal calf serum and supplemented as described (11) . The promyelocytic cell line HL-60 (ATCC CCL 240) was maintained in RPMI 1640 medium (Life Technologies, Inc.) containing 2 mM glutamine and 10% complement-inactivated fetal calf serum.

Purification of L-CanAg

Partially purified L-CanAg was prepared by trichloroacetic acid precipitation and Superose 6 gel filtration as described (12) . This procedure yielded the material used for HF treatment and immunization (see below). For peptide sequencing, the sample was further purified with affinity chromatography using immobilized Si-Le-reactive monoclonal antibody (mAb) C241 and anion exchange chromatography in 8 M urea, both as described (6) . Finally, the purified L-CanAg was passed over a 1-ml column of protein A-Sepharose (Pharmacia, Sollentuna, Sweden) to adsorb possible residual immunoglobulin from the affinity column; the flow-through fraction was then applied to a Superose 6 HR 10/30 gel filtration column (Pharmacia) and eluted in 0.1 M NHAc, pH 7.0. Fractions were tested for Si-Le content by fluoroimmunoassay. Fractions from the L-CanAg peak were collected and lyophilized. When finally purified, L-CanAg was subjected to peptide sequencing.

Peptide Sequencing

Highly purified L-CanAg corresponding to approximately 11 µg of protein was sequenced by Edman degradation (13) using an Applied Biosystems 477A pulsed liquid phase sequencer with on-line analysis of phenylthiohydantoin derivatives (14) .

Antibodies, Peptides, and DNA Probes

Partially purified L-CanAg was deglycosylated by HF treatment at room temperature overnight (15) and was used to immunize rabbits as described (16) . Approximately 5 µg of protein was used for each injection. The obtained antiserum will be referred to below as ``anti-L-CanAg/HF.''

The rabbit anti-CD43 antiserum (17) and the Si-Le-reactive mAb C241 (18) were gifts generously provided by Dr. Eileen Remold-O'Donnell (Center for Blood Research, Boston, MA) and CanAg Diagnostics AB (Göteborg, Sweden), respectively. The CD43-reactive mAb Leu-22 was purchased from Becton Dickinson (San José, CA). The plasmid pCD43TkHyg cfi containing CD43 cDNA was a kind gift from Drs. Jan Holgersson and Brian Seed (Harvard Medical School, Boston, MA). The CD43 insert was excised by digestion with NotI and HindIII and purified using the QIAEX DNA gel extraction kit (QIAGEN, Hilden, Germany).

Cell Labeling

COLO 205 cells or HL-60 cells were starved in methionine-free minimal essential medium (Life Technologies, Inc.) supplemented with 2 mM glutamine and 5% fetal calf serum 1 h prior to labeling. In some experiments, tunicamycin (Calbiochem, La Jolla, CA) was added to the methionine-free medium to a final concentration of 20 µg/ml (for COLO 205) or 5 µg/ml (for HL-60). Cells were labeled by the addition of a mixture of S-labeled methionine and cysteine (Pro-Mix, Amersham Corp.) in an amount corresponding to 200 µCi of [S]methionine/6-cm Petri dish. In pulse-chase experiments, the chase was initiated by the addition of unlabeled methionine and cysteine (both 100 µg/dish). The incorporation was stopped by putting the dishes on ice. Cells were washed twice with ice-cold 10 mM sodium phosphate, pH 7.2, 0.15 M NaCl and then lysed by brief sonication in 1.25 ml of the same buffer containing 50 mM Tris-HCl, pH 7.5, 5 mM EDTA, 1% Triton X-100, 0.5 mM phenylmethylsulfonyl fluoride, 1 µg/ml aprotinin, 25 µg/ml leupeptin, 0.7 µg/ml pepstatin, and 2 µg/ml calpain inhibitor I (the last two were from Boehringer Mannheim).

Immunoprecipitation

Labeled cell lysates were incubated overnight with 25 µl of nonimmune rabbit serum (NRS) at 4 °C followed by the addition of 150 µl of a 10% (w/v) suspension of fixed Staphylococcus aureus (Immunoprecipitin, Life Technologies, Inc.). After 30 min at room temperature, the Immunoprecipitin was removed by centrifugation at 14,000 g for 2 min. Precipitation with NRS was repeated once, and the Immunoprecipitin from the second NRS precipitation was saved and used as a nonspecific control. Specific antiserum (12 µl for anti-CD43, 25 µl for anti-L-CanAg/HF) was added and precipitated as described above. The Immunoprecipitin was washed 5 times with 1 ml of 10 mM Tris-HCl, pH 7.5, 2 mM EDTA, 0.1% Triton X-100, and 0.1% SDS and eluted by incubation at 95 °C with 50 mM Tris-HCl, pH 6.8, 10% glycerol, and 2% SDS.

SDS-PAGE and Autoradiography

Samples were applied to 1.5-mm-thick SDS-polyacrylamide gels (19) with 3% stacking gels and either 10% homogeneous or 3-15% gradient separation gels. C-Methylated Rainbow high molecular weight marker proteins (Amersham) were used as size references. Gels were fixed, dried, and exposed as described (6) .

Northern Blot Analysis

Total RNA was prepared from COLO 205 and HL-60 cells by the guanidinium thiocyanate-guanidinium chloride method (20) . Extraction of polyadenylated RNA and Northern blot analysis were performed as described (21, 22) . A Multi-Tissue Northern membrane (Clontech, Palo Alto, CA) was also probed with CD43 cDNA.


RESULTS

NH-terminal Peptide Sequence of L-CanAg

Seventeen rounds of Edman degradation of intact, highly purified L-CanAg yielded the sequence XXXAVQXPXXGEPLVXX, where X represents a cycle in which no amino acid could be identified. The glutamate in position 12 was regarded as ambiguous. A computer search of the EMBL data base using the TFASTA algorithm of the GCG program package (23) showed that the identifiable amino acids had a 100% identity with the NH-terminal of human CD43 (leukosialin, sialophorin) in its mature form (signal peptide removed). As shown in Fig. 1 , the unidentifiable positions in the L-CanAg sequence all coincided with Ser or Thr in the CD43 sequence. Because glycosylated amino acids are lost in the Edman analysis upon solvent extraction of the derivatized amino acid before chromatography, it seems likely that the blanks in the L-CanAg sequence were due to O-glycosylation of serines and threonines.


Figure 1: The NH-terminal amino acid sequence of L-CanAg, as determined by Edman degradation, compared with the CD43 sequence. Cycles in which no amino acid could be determined in the analysis of L-CanAg are marked by x. The lowercase e in position 12 of the L-CanAg sequence indicates that this identification was ambiguous. In the CD43 sequence, the site of cleavage of the hydrophobic signal peptide (26) is indicated by an arrow.



Detection of CD43 mRNA in COLO 205 Cells

Poly(A) RNA from the colon carcinoma cell line COLO 205, which produces L-CanAg, and from the promyelocytic cell line HL-60, which is known to produce CD43, was examined using a CD43 cDNA probe in a Northern blot hybridization. In both HL-60 and COLO 205, two hybridizing bands of 2.0 and 8.3 kilobases were found (Fig. 2). The amount of CD43 mRNA appeared to be substantially lower in COLO 205 than in HL-60, because the amounts of total RNA used were the same for both cell lines. None of the colon carcinoma cell lines SW1116, LoVo, or LS174T contained detectable levels of CD43 mRNA (data not shown).


Figure 2: Northern blot showing poly(A) RNA from the COLO 205 and HL-60 cell lines hybridized with a CD43 cDNA probe. Equal amounts of total RNA from COLO 205 and HL-60 were subjected to poly(A) RNA extraction and Northern blotting. The positions of RNA marker bands are indicated by the lines. kb, kilobases.



Immunoprecipitation of the L-CanAg Precursor Protein

Rabbit antisera against HF-deglycosylated L-CanAg (anti-L-CanAg/HF) and against CD43 were used in immunoprecipitation of cell lysates of metabolically labeled COLO 205 and HL-60 cells. In COLO 205, the anti-L-CanAg/HF antiserum and the anti-CD43 antiserum both precipitated a single band with an apparent molecular mass of 61 kDa (Fig. 3A). Upon tunicamycin treatment, the band precipitated by anti-L-CanAg/HF was shifted in apparent size to 58 kDa. Identical bands appeared in immunoprecipitation experiments with HL-60 cells, with both the anti-L-CanAg/HF (Fig. 3B) and the anti-CD43 antisera (data not shown). A pulse-chase experiment with COLO 205 cells showed that the anti-L-CanAg/HF band remained at its position until after 40 min of chase and then disappeared (Fig. 4). Similar results were obtained in HL-60 cells (data not shown).


Figure 3: Immunoprecipitation of lysates of metabolically labeled COLO 205 (A) and HL-60 cells (B) using antisera against deglycosylated L-CanAg and against CD43. Cells were starved for 1 h with (+) or without (-) tunicamycin (20 µg/ml for COLO 205; 5 µg/ml for HL-60) as indicated and labeled with 200 µCi of [S]methionine for 20 min. Lysates were precipitated twice with nonimmune rabbit serum (the second precipitate is shown in the lanes marked by NRS) before immunoprecipitation using antiserum against HF-deglycosylated L-CanAg (anti-L-CanAg/HF) or antiserum against CD43 (anti-CD43) (17). The samples were analyzed on a 10% SDS-PAGE gel. The lines indicate sizes in kDa of molecular mass marker proteins.




Figure 4: Immunoprecipitation of lysates of pulse-chase labeled COLO 205 cells using antiserum against deglycosylated L-CanAg. Cells were starved for 1 h, labeled for 10 min with 200 µCi of [S]methionine, and chased with excess unlabeled methionine for the time periods indicated. Lysates were precipitated twice with nonimmune rabbit serum (the second precipitate is shown in the lanes marked by NRS) before immunoprecipitation with antiserum against HF-deglycosylated L-CanAg (lanes marked by Ab). Samples were analyzed on a 3-15% gradient SDS-PAGE gel. The lines indicate sizes in kDa of molecular mass marker proteins.




DISCUSSION

L-CanAg is a relatively small (150-300 kDa) Si-Le-carrying mucin secreted by the colon adenocarcinoma cell line COLO 205. These cells also produce a membrane-bound MUC1 mucin, H-CanAg. In the present study, we have in several ways demonstrated that the apoprotein of L-CanAg is identical to at least a part of the apoprotein of the leukocyte-associated membrane sialoglycoprotein CD43. The amino-terminal peptide sequence of L-CanAg was identical to that of CD43 in all positions that could be determined (9 out of 17) (Fig. 1). Because the 8 unidentifiable positions in the L-CanAg sequence all corresponded to Ser or Thr in the CD43 sequence, it seems most likely that all of these hydroxy amino acids were glycosylated in L-CanAg, because glycosylated amino acids are not recovered in the sequencing procedure used. A similar NH-terminal glycosylation pattern has been reported for human plasma galactoglycoprotein, a fragment of CD43 present in normal blood (24) .

In order to confirm that the CD43 detected was indeed derived from the L-CanAg that was produced by the COLO 205 cells and not a result of some contamination of the samples, immunoprecipitation experiments were performed on lysates of metabolically labeled COLO 205 cells. Antiserum against HF-deglycosylated L-CanAg was found to precipitate a band with an apparent molecular mass of 61 kDa; a band of identical size was found with an antiserum against CD43. Identical results were obtained with both antisera in immunoprecipitation from lysates of the CD43-expressing promyelocytic cell line HL-60. The conclusion that the band that had precipitated with the anti-L-CanAg/HF antiserum was indeed the CD43 precursor protein was further strengthened by the shift in electrophoretic mobility upon tunicamycin treatment and the pulse-chase kinetics observed, because similar characteristics have been reported for the CD43 precursor (7, 17) . However, whereas the antisera against leukocyte CD43 also recognize the mature glycoprotein (apparent M approximately 115 kDa), which starts to appear after 10-20 min of chase in leukocyte cell lines (7, 17) , the reactivity of the anti-L-CanAg/HF antiserum seemed to be restricted to the nonglycosylated precursor both in COLO 205 and HL-60. It therefore seems likely that all epitopes recognized by the anti-L-CanAg/HF antiserum were directed to the extracellular, Ser/Thr-rich domain of the apoprotein and eventually became masked by O-glycosylation upon maturation of the glycoprotein. It should also be added that, although there is a wide discrepancy between the apparent M of the immunoprecipitated bands (61 kDa) and the molecular mass deduced from the cDNA sequence of CD43 (40 kDa), this highly aberrant electrophoretic mobility of the CD43 precursor has been observed in several other studies (17, 25) .

The expression of CD43 by the COLO 205 cells was also established by Northern blot hybridization, showing the two-transcript pattern typical of CD43 mRNA (26) both in COLO 205 and HL-60. Normal large and small intestine RNA were weakly hybridized in a Multi-Tissue Northern blot (data not shown); however, this result could be explained as a contribution from dispersed intestinal lymphatic tissue.

CD43 has hitherto been described as a membrane-bound sialoglycoprotein present on leukocytes and some other hematopoietic cells (9, 27) . Most of the present knowledge about the properties of CD43 derives from studies of leukocytes or leukocyte cell lines. Leukocyte CD43 carries 1 N-glycan and approximately 80 O-glycans on its extracellular NH-terminal portion, the latter mostly occurring as tetra- or hexasaccharides (9) . The 123-amino-acid cytoplasmic domain is constitutively phosphorylated and thought to be engaged in transmembrane signaling (28, 29, 30) . CD43 is proteolytically cleaved from the surface of leukocytes upon activation by binding of anti-CD43 mAbs (31, 32) . Released extracellular CD43 is found in normal blood as a ``plasma galactoglycoprotein'' at a concentration of >10 µg/ml (24, 33) .

L-CanAg shows several novel features when compared with the forms of CD43 that have been characterized earlier. First, although a few cases of binding of anti-CD43 mAbs to cancer cells of non-blood cell origin have been reported (34, 35) , this is the first unambiguous identification and characterization of a CD43 molecule expressed outside of the hematopoietic cell lineage. The three other colon carcinoma cell lines tested for CD43 expression in Northern blots and immunoprecipitation were all negative (data not shown). It is noteworthy that the cell line SW1116, which secretes a mucin-like molecule similar to L-CanAg in size, does not express CD43.

Second, in contrast to leukocyte CD43, which generally has been described as being released only upon activation, L-CanAg appears to be a constitutively secreted molecule. It seems likely that L-CanAg in fact consists only of the extracellular domain of CD43, released by proteolytic cleavage at the extracellular/luminal side of the molecule. The hypothesis that L-CanAg is a fragment of CD43 is supported by the observation that the anti-L-CanAg/HF antiserum seems to react only with the extracellular domain of CD43, indicating that other parts of the molecule were missing from the L-CanAg used for immunization. Moreover, the amino acid composition of L-CanAg matches that of extracellular CD43 more closely than that of total CD43 (6, 9) . A comparison of the amino acid yields in the peptide sequencing with the total protein amount analyzed also indicates that the apoprotein of L-CanAg cannot be as large as that of the entire CD43 (data not shown). It is not clear whether the putative cleavage event occurs intracellularly or at the cell surface. It is interesting to note that both the proteolytic cleavage of CD43 supposed to yield the plasma galactoglycoprotein (24) and the cleavage of the MUC1 mucin apoprotein, which occurs during biosynthesis (36) , have been tentatively located to extracellular Phe-Arg sequences close to the transmembrane domains of the respective glycoproteins.

Third, there is a major difference between L-CanAg and leukocyte CD43 concerning glycosylation. The predominant glycans of leukocyte CD43 have been described as disialylated tetra- or hexasaccharides (9) , depending on cell type. No individual glycan structure has been determined for L-CanAg, but carbohydrate analysis has shown that its sugar chains are considerably longer, with an average of about 17 sugars/chain, probably built on a polylactosamine backbone (6) . The abundance of fucose is also much higher in L-CanAg (14% of total sugars) than in leukocyte CD43 (1.2% in HL-60 cells (9) ), as is the total carbohydrate content (85% (w/w) in L-CanAg, 67% in CD43 from HL-60 cells (9) ). The higher degree of glycosylation of L-CanAg also explains its higher molecular mass as estimated by SDS-PAGE (150-300 kDa (6) ) when compared with leukocyte CD43 (115-135 kDa (27) ). It has previously been shown that different glycoforms of CD43 have markedly different mobilities in SDS-PAGE (37) .

Given all these differences between leukocyte CD43 and L-CanAg, it does not seem surprising that the type of CD43 expression displayed by COLO 205 cells has escaped notice until now. The standard method for detecting CD43 has been tissue immunostaining or flow cytometry using glycosylation-dependent mAbs raised against leukocyte glycoforms of CD43. One such mAb, Leu-22, was tested for binding to L-CanAg in fluoroimmunoassay and showed no reactivity (data not shown). This suggests the possibility that, if other carcinomas also express L-CanAg-like CD43 glycoforms, they have not been possible to detect with the anti-CD43 mAbs commonly used.

One might speculate over the possible functional significance of CD43 expression in carcinoma cells. CD43 has been described as being an anti-adhesion molecule that prevents interactions between leukocytes (10, 38). The expression of CD43 by a carcinoma cell might therefore facilitate tumor dissemination. The observation that HeLa cells experience decreased T-cell adhesion upon transfection with CD43 (39) strongly suggests that CD43 expression may help malignant cells to evade immune recognition. Recently, it has also been shown that L-CanAg can cause Si-Le-dependent inhibition of leukocyte binding to E-selectin, an endothelial leukocyte receptor (12). Secretion of Si-Le- or Si-Le-carrying glycoforms of CD43 may therefore be advantageous to cancer cells by impeding leukocyte recruitment to the tumor. The cell-activating properties of CD43, which in some cases lead to proliferation (29) , might also influence the phenotype of a CD43-expressing carcinoma cell.


FOOTNOTES

*
This work was supported by funds from the Swedish Cancer Fund, by Grants 7461 and 10443 from the Swedish Medical Research Council, and by funds from Ingabritt and Arne Lundberg's Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The abbreviations used are: Si-Le, sialyl-Lewis blood group antigen; mAb, monoclonal antibody; NRS, nonimmune rabbit serum; PAGE, polyacrylamide gel electrophoresis.


ACKNOWLEDGEMENTS

We express our gratitude to CanAg Diagnostics AB for providing monoclonal antibodies, to Dr. Eileen Remold-O'Donnell for the gift of the anti-CD43 antiserum and for helpful suggestions, and to Drs. Brian Seed and Jan Holgersson for sending the plasmid containing CD43 cDNA.


REFERENCES
  1. Strous, G. J., and Dekker, J.(1992) Crit. Rev. Biochem. Mol. Biol. 27, 57-92 [Abstract]
  2. Carlstedt, I., Sheehan, J. K., Corfield, A. P., and Gallagher, J. T. (1985) Essays Biochem. 20, 40-76 [Medline] [Order article via Infotrieve]
  3. Hilkens, J., Ligtenberg, M. J. L., Vos, H. L., and Litvinov, S. V. (1992) Trends Biochem. Sci. 17, 359-363 [CrossRef][Medline] [Order article via Infotrieve]
  4. Sako, D., Chang, X.-J., Barone, K. M., Vachino, G., White, H. M., Shaw, G., Veldman, G. M., Bean, K. M., Ahern, T. J., Furie, B., Cumming, D. A., and Larsen, G. R.(1993) Cell 75, 1179-1186 [Medline] [Order article via Infotrieve]
  5. Carraway, K. L., Fregien, N., Carraway, K. L., III, and Carothers, C. A.(1992) J. Cell Sci. 103, 299-307 [Free Full Text]
  6. Baeckström, D., Hansson, G. C., Nilsson, O., Johansson, C., Gendler, S. J., and Lindholm, L.(1991) J. Biol. Chem. 266, 21537-21547 [Abstract/Free Full Text]
  7. Carlsson, S. R., and Fukuda, M.(1986) J. Biol. Chem. 261, 12779-12786 [Abstract/Free Full Text]
  8. Remold-O'Donnell, E., Davis, A. E., III, Kenney, D., Bhaskar, K. R., and Rosen, F. S.(1986) J. Biol. Chem. 261, 7526-7530 [Abstract/Free Full Text]
  9. Fukuda, M.(1991) Glycobiology 1, 347-356 [Abstract]
  10. Remold-O'Donnell, E., and Parent, D.(1994) J. Immunol. 152, 3595-3605 [Abstract/Free Full Text]
  11. Larson, L. N., Johansson, C., Lindholm, L., and Holmgren, J.(1988) Int. J. Cancer 42, 877-882 [Medline] [Order article via Infotrieve]
  12. Zhang, K., Baeckström, D., and Hansson, G. C.(1994) Int. J. Cancer 59, 823-829 [Medline] [Order article via Infotrieve]
  13. Edman, P., and Begg, G.(1967) Eur. J. Biochem. 1, 80-91 [Medline] [Order article via Infotrieve]
  14. Hewick, R. M., Hunkapiller, M. W., Hood, L. E., and Dreyer, W. J. (1981) J. Biol. Chem. 256, 7990-7997 [Abstract/Free Full Text]
  15. Baeckström, D., Karlsson, N., and Hansson, G. C.(1994) J. Biol. Chem. 269, 14430-14437 [Abstract/Free Full Text]
  16. Hansson, G. C., Baeckström, D., Carlstedt, I., and Klinga-Levan, K.(1994) Biochem. Biophys. Res. Commun. 198, 181-190 [CrossRef][Medline] [Order article via Infotrieve]
  17. Remold-O'Donnell, E., Kenney, D., and Rosen, F. S.(1987) Biochemistry 26, 3908-3913 [Medline] [Order article via Infotrieve]
  18. Johansson, C., Nilsson, O., Baeckström, D., Jansson, E.-L., and Lindholm, L.(1991) Tumor Biol. 12, 159-170
  19. Laemmli, U. K.(1970) Nature 227, 680-685 [Medline] [Order article via Infotrieve]
  20. MacDonald, R. J., Swift, G. H., Przybyla, A. E., and Chirgwin, J. M. (1987) Methods Enzymol. 152, 219-227 [Medline] [Order article via Infotrieve]
  21. Baeckström, D., Nilsson, O., Price, M. R., Lindholm, L., and Hansson, G. C.(1993) Cancer Res. 53, 755-761 [Abstract]
  22. Gum, J. R., Kam, W. K., Byrd, J. C., Hicks, J. W., Sleisenger, M. H., and Kim, Y. S.(1987) J. Biol. Chem. 262, 1092-1097 [Abstract/Free Full Text]
  23. Program Manual for the Wisconsin Package(1994) Version 8, Genetics Computer Group, Madison, WI
  24. Schmid, K., Heidegger, M. A., Brossmer, R., Collins, J. H., Haupt, H., Marti, T., Offner, G. D., Schaller, J., Takagaki, K., Walsh, M. T., Schwick, H. G., Rosen, F. S., and Remold-O'Donnell, E.(1992) Proc. Natl. Acad. Sci. U. S. A. 89, 663-667 [Abstract]
  25. Shelley, C. S., Remold-O'Donnell, E., Davis, A. E., III, Bruns, G. A. P., Rosen, F. S., Carroll, M. C., and Whitehead, A. S.(1989) Proc. Natl. Acad. Sci. U. S. A. 86, 2819-2823 [Abstract]
  26. Pallant, A., Eskenazi, A., Mattei, M.-G., Fournier, R. E. K., Carlsson, S. R., Fukuda, M., and Frelinger, J. G.(1989) Proc. Natl. Acad. Sci. U. S. A. 86, 1328-1332 [Abstract]
  27. Remold-O'Donnell, E., and Rosen, F. S.(1990) Immunodefic. Rev. 2, 151-174 [Medline] [Order article via Infotrieve]
  28. Axelsson, B., and Perlmann, P.(1989) Scand. J. Immunol. 30, 539-547 [Medline] [Order article via Infotrieve]
  29. Silverman, L. B., Wong, R. C. K., Remold-O'Donnell, E., Vercelli, D., Sancho, J., Terhorst, C., Rosen, F., Geha, R., and Chatila, T.(1989) J. Immunol. 142, 4194-4200 [Abstract/Free Full Text]
  30. Wong, R. C. K., Remold-O'Donnell, E., Vercelli, D., Sancho, J., Terhorst, C., Rosen, F., Geha, R., and Chatila, T.(1990) J. Immunol. 144, 1455-1460 [Abstract/Free Full Text]
  31. Bazil, V., and Strominger, J. L.(1993) Proc. Natl. Acad. Sci. U. S. A. 90, 3792-3796 [Abstract]
  32. Rieu, P., Porteu, F., Bessou, G., Lesavre, P., and Halbwachs-Mecarelli, L.(1992) Eur. J. Immunol. 22, 3021-3026 [Medline] [Order article via Infotrieve]
  33. Schmid, K., Mao, S. K. Y., Kimura, A., Hayashi, S., and Binette, J. P. (1980) J. Biol. Chem. 255, 3221-3226 [Abstract/Free Full Text]
  34. Krenova, D., Otova, B., and Kren, V.(1993) Transplant. Proc. 25, 2797-2798 [Medline] [Order article via Infotrieve]
  35. Ioachim, H. L., Pambuccian, S., Giancotti, F., and Dorsett, B.(1994) Int. J. Cancer Suppl. 8, 132-133
  36. Ligtenberg, M. J. L., Kruijshaar, L., Buijs, F., van Meijer, M., Litvinov, S. V., and Hilkens, J.(1992) J. Biol. Chem. 267, 6171-6177 [Abstract/Free Full Text]
  37. Maemura, K., and Fukuda, M.(1992) J. Biol. Chem. 267, 24379-24386 [Abstract/Free Full Text]
  38. Manjunath, N., Johnson, R. S., Staunton, D. E., Pasqualini, R., and Ardman, B.(1993) J. Immunol. 151, 1528-1534 [Abstract/Free Full Text]
  39. Ardman, B., Sikorski, M. A., and Staunton, D. E.(1992) Proc. Natl. Acad. Sci. U. S. A. 89, 5001-5005 [Abstract]

©1995 by The American Society for Biochemistry and Molecular Biology, Inc.