2Departamento de Bioquímica, Instituto Nacional de Enfermedades Respiratorias, Calzada de Tlalpan 4502, 01040 México, 3Laboratorio de Inmunología, Departamento de Bioquímica, Facultad de Medicina, UNAM, 04510, México, and 4Laboratoire de Chimie Biologique de la Université des Sciences et Technologies de Lille, UMR du CNRS n° 8576, Villeneuve dAscq, 59655 France
Received June 28, 1999; revised on October 27, 1999; accepted on October 31, 1999.
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Abstract |
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Key words: thymocyte/glycoproteins/lectins/Amaranthus leucocarpus/ontogeny
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Introduction |
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Lectins are excellent tools for oligosaccharide characterization as well as for isolation of cellular populations. Lectins, which show specificity for O-glycosidically linked glycans, have been widely used in the fractionation of thymocytes and lymphocyte subpopulations. By selective agglutination with peanut agglutinin (Arachis hypogaea), it is possible to purify cortical immature thymocytes (Reisner et al., 1976a). Using the same procedure, T and B splenocytes were fractionated with the lectin from Glycine max (Reisner et al., 1976b
). Helix pomatia can be employed for the identification and isolation of T cells (De Petris and Tackacs, 1983
), and Vicia villosa agglutinin recognizes specifically lymphocytes bearing the CD8+ (cytotoxic) phenotype (Fortune and Lehner, 1988
). Other lectins such as wheat germ agglutinin, specific for GlcNAc have been used in the purification of B lymphocytes (De Dios et al., 1986
). Sequential fractionation of lymphocytes by soybean and peanut agglutinins yielded a pluripotential stem cells enriched fraction devoid of graft versus host activity, which has been successfully transplanted into patients with severe immune deficiencies (Reisner, 1983
). Moreover, some of these lectins are currently used to evaluate the immune status of patients (Sharon, 1983
). In previous works we demonstrated that the lectin from Amaranthus leucocarpus (ALL) possesses the capacity to interact with murine medullary thymocytes (Lascurain et al., 1994
), murine nonactivated peritoneal macrophages (Gorocica et al., 1998
; Maldonado et al., 1998
), and human naive T-lymphocytes (Lascurain et al., 1997
). ALL is a 35 kDa glycoprotein specific for the T and the Tn antigens (Gal ß1,3GalNAc
1,O-Ser/Thr and GalNAc
1,O-Ser/Thr, respectively) (Zenteno et al., 1992
). This lectin agglutinates preferentially erythrocytes with the M phenotype, does not recognize B lymphocytes, shows low mitogenic activity on human lymphocytes (Lascurain et al., 1997
), and induces suppression in mice (Zenteno et al., 1985
). Although it has been reported that all the cells recognized by ALL share the characteristic of being naive or quiescent cells (Lascurain et al., 1997
; Gorocica et al., 1998
), until now the specific role of the lymphocyte subset recognized by this lectin has not been elucidated. This work provides information on the molecular characteristics of the ALL receptor from murine medullary thymocytes.
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Results |
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Discussion |
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The thymocyte receptor for ALL is a glycoprotein of 70 kDa, which seems to be made up of three isoforms that can be separated by ion exchange chromatography. The ALL-thymocyte receptor contains mainly glutamic, aspartic, serine, proline, and glycine residues. Its glycans contain mainly Gal, GalNAc, and NeuAc residues as typical sialylated O-glycosylproteins. But the presence of mannose and GlcNAc residues indicates that the receptor contains also N-glycosidically linked glycans. The amino acid composition of the three isoforms is almost identical to that of the affinity chromatography purified receptor. The main differences among these fractions were observed in their sugar concentration and degree of sialylation; the isoform ALLTr3 is the most sialylated, suggesting that, as in other membrane O-glycosylproteins, the glycosylation state is modified according to the activation state of the cell (Carlsson and Fukuda, 1986; Piller et al., 1988
).
By means of lectins with similar specificity to ALL, several authors characterized different leukocyte antigens; PNA recognizes a major glycoprotein of 170180 kDa and minor bands of 110120 kDa (De Maio et al., 1986); the lectin from Salvia sclarea interacts specifically with a 125 kDa glycoprotein that corresponds to leukosialin (CD43 or sialophorin) (Piller et al., 1988
). Mucin-like structures have been identified in other leukocyte antigens (Shimizu and Shaw, 1993
), such as CD45 (95 kDa) and Ly5 or T 200 (210 kDa). Although the potential receptor for ALL has been assumed to be leukosialin (Figure 4), which is the major carrier of O-glycosidically linked glycans in lymphocytes (Carlsson and Fukuda, 1986
; Shelley et al., 1989
), no cross reaction was identified either between the ALL receptor and known phenotypic markers, or with leukosialin (Figure 4). These results were confirmed by analysis of the amino acid sequence of the ALL-thymocyte receptor. The receptor and its isoforms have blocked N-terminal amino acid residue; the analysis of tryptic peptides from the receptor by MALDI-TOF indicated that the protein shows low (<17%) homology with proteins such as the human cerebral KIAA0659 protein (Ishikawa et al., 1998
), Fas-associated death domain protein (Fernandes-Alnemri et al., 1996
), and transforming growth factor-ß type II receptor (Suzuki et al., 1994
).
ALL interacts also with resident murine peritoneal macrophages through a 68 kDa receptor, its amino acid composition being different from that of the thymocyte receptor (Gorocica et al., 1998), indicating the presence of common protein motifs containing O-glycosidically linked glycans in cells derived from lymphoid and myeloid cell lineage. Although the function of the ALL receptor remains to be identified, our results suggest that the ALL receptor could be considered a novel molecular marker for naive or quiescent T-cell populations.
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Materials and methods |
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Cells
Thymus glands were surgically removed from male CD-1 mice (4 weeks old), washed twice in phosphate-buffered saline (PBS: 0.15 M NaCl, 0.05 M sodium phosphate, pH 7.2) and passed through a fine mesh to harvest the suspended cells. Thymocytes were suspended in Dulbeccos modified Eagles medium supplemented with 5% fetal calf serum (heat inactivated at 56°C for 30 min) at 108 cells/ml. The ALL+ thymocyte subpopulation was purified by selective agglutination as follows: 1 ml of the cell suspension was incubated for 10 min at room temperature with 10 µg of biotinylated-ALL, then the cell suspension was layered gently on top of a 7% bovine serum albumin solution in PBS in conical 12 ml glass tubes. After 30 min at room temperature, the cells present in the bottom (ALL+ thymocytes) and on the top (ALL- thymocytes) were collected separately and washed twice with PBS (Reisner, 1983).
Analysis of separated cells
Viability of purified thymocytes (>90%) was assessed by the trypan blue exclusion test. Phenotypic characterization of the ALL+ cell fractions was determined by the double stain immunological method (Hudson and Hay, 1980). ALL+ cells were incubated at room temperature for 15 min with 10 µl FITC anti-CD8 diluted 1:150/106 cells in 500 µl PBS-BSA-azide (PBS with 0.2% bovine serum albumin and 0.2% sodium azide) and 10 µl PE-labeled anti-CD2, CD3, or CD4, diluted 1:200, as the second color. CD45RB was determined on ALL+ thymocytes using a single fluorescence assay using 10 µl of FITC-anti CD45RB (diluted 1:150/106 cells in PBS-BSA-azide). After incubation, the cells were washed with PBS-BSA and suspended in 500 µl of PBS with 0.1% p-formaldehyde; fluorescent cells were determined by flow cytometry in an Excalibur Becton & Dickinson Cell Sorter apparatus (FACs, Mountain View, CA). In all cases the biotin-labeled ALL was present, and no variations in the phenotypic characterization were observed if the lectin was eliminated previously by addition of 0.2 M GalNAc. Control staining was performed using FITC-labeled antibodies against ALL.
Receptor purification
Indirect affinity chromatography was used as a method to purify the lectin-binding glycoproteins from the thymocyte (Buckie and Cook, 1986); 108 thymocytes purified by agglutination with biotin-ALL were lysed in a solution of PBS containing 1 µg/ml aprotinin A, 1 µg/ml pepstatin, 2 µg/ml leupeptin, 2 mM phenylmethylsulfonyl fluoride, and 0.1% (v/v) Triton X-100 (lysis buffer), for 30 min at 4°C under shaking. Nuclei, cell debris, and mitochondria were removed by centrifugation, first for 10 min at 250 x g, then 30 min at 18,000 x g. Pellets were eliminated and the clear supernatant was loaded on an avidin-agarose column (3 x 1 cm), equilibrated previously with PBS-T (PBS containing 0.1% v/v Triton X-100) at 4°C. The unretained material was eluted with PBS-T and the fraction corresponding to the bound protein was eluted with 0.2 M GalNAc in PBS-T, and the biotin-labeled lectin was eluted from the avidin column by addition of 0.2 M glycine/HCl, pH 2.8. Optical density A280 was determined on fractions dialyzed against PBS. Finally, the GalNAc-eluted fractions were pooled, dialyzed against distilled water, and freeze-dried for further analysis. In order to avoid nonspecific interaction among the avidin column and cell proteins, the thymocyte lysate in absence of biotin-labeled lectin or with unlabeled lectin was deposed onto the column. Our results indicate that, under these conditions, almost all deposed protein was recovered in the unretained fraction and no protein was detected in fractions eluted with either GalNAc or O.2 M glycine/HCl pH 2.8.
Separation of ALL+ thymocyte receptor isoforms
The affinity purified thymocyte receptor was applied to a mono P prepacked HR column 5/5 mm (Pharmacia, Uppsala, Sweden) equilibrated previously with 50 mM Bis-Tris buffer, pH 7.5, at a flow rate of 1 ml/min with a maximal pressure of 40 bars, in a 60 min program using an FPLC system (Pharmacia, Uppsala, Sweden). Thymocyte receptor isoforms were eluted from the column with a 01 M NaCl stepwise gradient in Bis-Tris buffer. Fractions of 1 ml were collected and optical density was monitored at A280. Each eluted peak was dialyzed against distilled water before lyophilization for further analysis.
Polyacrylamide gel electrophoresis
The molecular mass and the homogeneity of the purified receptor were evaluated by polyacrylamide gel electrophoresis (PAGE) in the presence of 0.1% sodium dodecyl sulfate (SDS), using the Laemmli (1970) buffer system; the gels were stained with 0.1% Coomassie brilliant blue.
Analytical methods
Protein concentration was determined by the method of Lowry modified by Peterson (1977), using bovine serum albumin as standard. Carbohydrate concentration was determined by the method of Dubois et al. (1956)
, using lactose as standard. Carbohydrate composition analysis was performed by methanolysis in the presence of meso-inositol as internal standard; the per-O-trimethyl silylated methyl glycosides (after N-re-acetylation) were analyzed by gas-chromatography using a capillary column (25 x 0.32 mm) of 5% Silicone OV 210 (Applied Science Lab., Buffalo, NY), in a Varian 2100 gas chromatograph (Orsay, France; Zanetta et al., 1972
).
Amino acid analysis
A 100 µg sample was hydrolyzed under vacuum with 2 ml of 6 M HCl at 110°C in sealed tubes for 24, 48, and 72 h. The samples were analyzed on an automatic amino acid analyzer Durrum 500, according to Bidlingmeyer et al. (1984), using Nor-leucine as internal standard. The amino acid sequence analysis was determined in samples of purified ALL-thymocyte receptor and isoforms. Samples were separated by SDSPAGE and electroblotted on a PVDF membrane; the band was excised from the blot and sequenced with a Beckman Model LF3000 protein sequencer (Fullerton, CA). Amino acid sequencing determined by MALDI-TOF on peptide fragments obtained by trypsin digestion of the purified ALL-receptor was performed on the SDSPAGE excised band as follows: the gel containing 200 pM of receptor was digested with 0.5 µg trypsin in 500 µl ammonium bicarbonate, pH 8.0 at 37°C, for 24 h. The reaction was inhibited by storing at 4°C. Then, the enzyme digest was evaporated to dryness using a Gyrovap (Howe, London). Samples were prepared by mixing directly onto the target 1 µl of the reaction products (containing 50 pM) and 1 µl of a 2,5-dihydroxibenzoic acid matrix (12 mg/ml in CH3OH /H20, 70:30, v/v), and then allowing the mixture to crystallize at room temperature. Positive ions of the peptides were measured by MALDI-TOF on a Vision 2000 time-of-flight mass spectrometer (Finnigan MAT, Bremen, Germany) equipped with a 337 nm UV laser. The mass spectra were acquired in reflectron mode under 8 keV acceleration voltage and positive detection. Control assays were performed using trypsin alone to identify self-digested peptide mass and with angiotensin I as standard (Mr 1296.7). The mass of [M+H]+ ions from peptides produced by tryptic digestion was compared with those obtained from NCBInr (Swiss-Prot 10/01/99) data base (Hellman et al., 1995
).
Protein blotting
Thymocytes (2 x 106) were solubilized in lysis buffer. Insoluble material was removed by centrifugation first for 10 min at 250 x g, then 30 min at 18,000 x g and detergent soluble proteins were resolved by 10% SDSpolyacrylamide gel electrophoresis (PAGE). Resolved proteins were transferred to nitrocellulose membranes by using a semidry blotting apparatus (Bio-Rad, Richmond, CA) under conditions recommended by the manufacturer. Membranes containing transferred proteins were blocked overnight with TBS (Tris-base 20 mM, NaCl 137 mM, pH 7.6, and 0.1% Tween 20) and 5% skimmed milk, prior to incubation with antibodies against the CD43 isoforms S7 and 1B11 (anti-115 kDa and anti-130 kDa, respectively). Membranes were then washed in TBS and primary antibodies were detected with rat anti-mouse antibodies conjugated with horseradish peroxidase and an ECL (Enhanced chemiluminescence) detection system. To evaluate the binding of ALL to murine thymocyte glycoproteins and to the purified receptor, these proteins were transferred to nitrocellulose membranes. Membranes containing resolved proteins were blocked overnight in TBS containing 5% skimmed milk and incubated with ALL-biotin diluted in PBS and with 5% skimmed milk for 1 h at 37°C and overnight at 4°C. The blot was then washed with TBS without Tween-20 and incubated with Extravidin®-peroxidase. Proteins were visualized by ECL. Negative controls were performed using biotin-labeled antibodies raised against the purified lectin and revealed no interaction with the purified thymocyte receptor or with the total lysate.
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Acknowledgments |
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Footnotes |
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