Isolation of the receptor for Amaranthus leucocarpus lectin from murine naive thymocytes

Flor Porras2, Ricardo Lascurain3, Raúl Chávez3, Blanca Ortiz2, Pedro Hernández2, Henri Debray4 and Edgar Zenteno1,3

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 d’Ascq, 59655 France

Received June 28, 1999; revised on October 27, 1999; accepted on October 31, 1999.


    Abstract
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 References
 
From murine medullary thymocytes we purified the receptor for the Amaranthus leucocarpus lectin (ALL) using a complex with the biotin-labeled lectin and avidin-agarose as the affinity matrix. Most ALL+ thymocytes (83%) are naive cells with the CD4+CD8-CD45RB+ phenotype. The receptor for this lectin is a 70 kDa glycoprotein that contains 20% of sugar by mass. It is constituted mainly by aspartic and glutamic acids, serine, proline, and glycine; its glycosidic portion contains mainly O-glycosidically linked glycans with Gal, GalNAc and NeuAc residues as well as one N-glycosidically linked glycan per molecule. Ionic strength chromatography revealed that the ALL-thymocyte receptor (ALLTr) is made up by three isoforms, which possess similar amino acid composition but show slight differences in their sugar composition. The N-terminal amino acid residues are blocked both in the receptor and its purified isoforms. Analyses of the receptor’s peptides, obtained by trypsin digestion with MALDI-TOF (matrix assisted laser desorption ionization-time of flight), were compared with the relative values obtained from the NCBInr (Swiss-Prot 10/01/99) database. Our results indicate that the peptides of ALLTr show low homology (<17%) with the human KIIA protein, the Fas-associated death domain protein, and the transforming growth factor-ß type II receptor. Our results suggest that the ALL thymocyte receptor could be considered a novel phenotypic marker specific for naive T cells.

Key words: thymocyte/glycoproteins/lectins/Amaranthus leucocarpus/ontogeny


    Introduction
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 References
 
Bone marrow–derived nucleated cells are known to possess glycoproteins containing O-glycosidically glycans linked through N-acetyl-D-galactosamine (GalNAc) to hydroxyl groups of serine or threonine residues. These O-glycosyl­proteins or mucin-like glycoproteins appear to be of different sizes, depending on the state and the type of cells. Previous works indicate that the mucin-like structures are specific for each cell lineage and for different differentiation stages, within a given cell lineage (Fukuda, 1992Go; Muroi et al., 1997Go). For these reasons, it is of particular interest to identify specific tools for the study of the specific role of O-glycans in cell physiology.

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., 1976aGo). Using the same procedure, T and B splenocytes were fractionated with the lectin from Glycine max (Reisner et al., 1976bGo). Helix pomatia can be employed for the identification and isolation of T cells (De Petris and Tackacs, 1983Go), and Vicia villosa agglutinin recognizes specifically lymphocytes bearing the CD8+ (cytotoxic) phenotype (Fortune and Lehner, 1988Go). Other lectins such as wheat germ agglutinin, specific for GlcNAc have been used in the purification of B lymphocytes (De Dios et al., 1986Go). 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, 1983Go). Moreover, some of these lectins are currently used to evaluate the immune status of patients (Sharon, 1983Go). In previous works we demonstrated that the lectin from Amaranthus leucocarpus (ALL) possesses the capacity to interact with murine medullary thymocytes (Lascurain et al., 1994Go), murine nonactivated peritoneal macrophages (Gorocica et al., 1998Go; Maldonado et al., 1998Go), and human naive T-lymphocytes (Lascurain et al., 1997Go). ALL is a 35 kDa glycoprotein specific for the T and the Tn antigens (Gal ß1,3GalNAc {alpha}1,O-Ser/Thr and GalNAc{alpha}1,O-Ser/Thr, respectively) (Zenteno et al., 1992Go). This lectin agglutinates preferentially erythrocytes with the M phenotype, does not recognize B lymphocytes, shows low mitogenic activity on human lymphocytes (Lascurain et al., 1997Go), and induces suppression in mice (Zenteno et al., 1985Go). Although it has been reported that all the cells recognized by ALL share the characteristic of being naive or quiescent cells (Lascurain et al., 1997Go; Gorocica et al., 1998Go), 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.


    Results
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 References
 
Cellular purification
The thymocytes agglutinated by biotin-labeled ALL (ALL+) corresponded to 5% (0.3) of the total thymocytes. ALL+ cells bear mainly the mature thymocyte phenotypes, CD2+CD3+CD4+CD8- (83.4%), 14.8% of the cells are CD2+CD3+CD4+CD8+, 0.4% are CD2+CD3+CD4-CD8+, and 1.3% are CD2+CD3+CD4-CD8- (Figure 1a). Almost 90% of the ALL-purified cells possess also the CD45RB+ phenotype (Figure 1b); control experiments indicated that ~8% of the total thymocytes corresponded to naive CD45RB+ cells.



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Fig. 1. Phenotypic characterization of murine thymocytes purified by selective agglutination with biotin-labeled Amaranthus leucocarpus lectin. (a) Analyses were performed using PE labeled anti-CD4 and FITC-labeled anti-CD8 as second color. (b) FITC-labeled anti-CD45RB was used to identify thymocytes with naive phenotype. Almost 90% of the ALL-purified cells are CD45RB+ (Clear) and ~8% of the total thymocytes corresponded to naive CD45RB+ cells (Dark).

 
Receptor purification
From ~108 thymocytes, 27.4 mg of soluble protein were obtained after lysis. The receptor for ALL was purified in a single step from the thymocyte lysate by an indirect affinity chromatography method, using biotin-labeled ALL and avidin–agarose as affinity support. The receptor was eluted from the affinity matrix specifically by adding 0.2 M GalNAc (Figure 2). The purified protein corresponds to 180 µg (<1%) of the proteins from the cell lysate.



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Fig. 2. Purification of the murine thymocyte receptor for Amaranthus leucocarpus lectin. The cells prior to lysis were incubated with biotin-labeled ALL, and then the complex was purified on an avidin–agarose column. The unretained fraction was eluted with PBS-T (0.1% Triton X-100) and the affinity purified receptor was eluted by addition of 0.2 M GalNAc in PBS-T. Optical density A280 was determined on fractions dialyzed previously against PBS.

 
Polyacrylamide gel electrophoresis and blotting
SDS–PAGE analysis of the purified ALL-receptor from murine thymocytes showed that the purified fraction is homogeneous, giving a single band of 70 kDa (Figure 3). Experiments using ALL as control indicated a band of a 35 kDa protein, confirming that the 70 kDa band obtained by affinity chromatography corresponds to the purified receptor (Figure 3). Blotting of murine thymocytes lysate was revealed with antibodies against the CD43 isoforms S7 (which recognize the 115 kDa isoform) and 1B11 (130 kDa), and with biotin labeled ALL. Our results indicate that the antibodies against CD43 isoforms, recognized in the thymocytes extract, a single band, which corresponds to its specific proteins, (Figure 4, lanes 1, 2), and ALL recognizes only a 70 kDa protein in the same cell lysate (Figure 4, lane 3). The purified ALL thymocyte receptor is recognized by the lectin (Figure 4, lane 4).



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Fig. 3. SDS–PAGE of the purified ALL-thymocyte receptor. Lane A, 50 µg of thymocyte lysate. Lane B, 10 µg of purified fraction eluted with 0.2 M GalNAc. Lane C, 10 µg Amaranthus leucocarpus lectin. The molecular weight markers are: myosin (205 kDa), phosphorylase b (94 kDa), bovine serum albumin (67 kDa), ovalbumin (45 kDa), carbonic anhydrase (30 kDa), trypsin inhibitor (20.1 kDa) and {alpha}{angle}lactalbumin (14.4 kDa).

 


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Fig. 4. Immunoblot of murine thymocytes and purified ALL-thymocyte receptor. Thymocyte lysates (from 2 x 106 cells) were electrophoresed in SDS–polyacrylamide gel electrophoresis, transferred to nitrocellulose filters, and analyzed with S7 (anti-115 kDa isoform of murine CD43, lane 1), and 1B11 (anti-CD43, 130 kDa isoform, lane 2). Cell lysate (lane 3) and the affinity chromatography purified receptor (lane 4) were analyzed with biotin-labeled ALL. Lanes 1 and 2 were revealed with rat anti-mouse conjugated with horseradish peroxidase, and lanes 3 and 4 with Extavidin–peroxidase in an enhanced chemiluminescence detection system.

 
Purification of ALL thymocyte receptor isoforms
From the affinity purified ALL receptor we obtained three isoforms by ion exchange chromatography on a mono P column, in anionic form. The isoforms (termed as ALLTr1, ALLTr2, and ALLTr3) were eluted with a stepwise gradient of NaCl. The amount of protein obtained in each fraction indicated that the most important isoform is the ALLTr1, which corresponds to 50% of the receptor applied on the column; ALLTr2 and ALLTr3 corresponded to 25% and 15% of the protein applied on the mono P column (Figure 5).



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Fig. 5. Purification of murine thymocyte receptor (ALLTr)-isoforms for Amaranthus leucocarpus lectin by ion exchange chromatography on a mono P column (anionic form) in an FPLC system. The affinity purified ALL receptor (500 µg) was applied to the column equilibrated previously with 50 mM Bis-Tris buffer pH 7.5. Isoforms were eluted by a stepwise NaCl gradient (dotted line). Detection of the optical density of each 1 ml fraction was at A280 (continuous line).

 
Chemical characterization
The thymocyte receptor for ALL is a glycoprotein, with 20% of sugars by weight, containing mainly aspartic, glutamic, serine, proline, and glycine residues; no specific peak of cysteic acid was found after performic acid oxidation (Table I). The carbohydrate fraction of the receptor, which represents 20% by weight, contains mainly N-acetyl-D-galactosamine and galactose, but N-acetyl-D-glucosamine, mannose, and sialic acid are also present in smaller amounts (Table II). The analysis of the purified isoforms indicated slight differences in the concentration of carbohydrates by weight, and quantitative differences in the concentration of sialic acid; as indicated in Table II the most sialylated isoform is represented by the ALLTr3 fraction. The receptor and its purified isoforms possess blocked N-terminal amino acid residues. Tryptic digestion of the purified receptor, analyzed by MALDI-TOF, yields 25 peptidic fractions, the m/z of the identified fractions ranged from 442.9 to 3817.2, but the main fractions were located at 689.9, 1284.7, 1480.7, 1957.2, 2251.1, and 2809.1. The molecular [M+H]+ ions from the MALDI-TOF spectrum of tryptic digested thymocyte receptors were compared with those obtained from the NCBInr (Swiss-Prot 10/01/99) database. The identified thymocyte peptides showed 17% homology with the KIAA0659 protein, which is a predicted coding sequence obtained from cDNA clones from brain, 10% with the transforming growth factor-ß type II receptor, and 7% with Fas-associated death domain protein interleukin-1b-converting enzyme.


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Table I. Amino acid composition of the thymocyte-receptor for Amaranthus leucocarpus lectin
 

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Table II. Carbohydrate composition of ALL-thymocyte receptor and isoforms
 

    Discussion
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 References
 
T lymphocytes differentiate within the thymus along the CD4/CD8 developmental pathway in a highly ordered process (Sprent, 1989Go). Maturation of thymocytes is assessed by expression of the T cell receptor and the CD4 and CD8. The leukocyte common antigen, CD45, has been promoted as a potential marker of memory T cells. Resting or naive CD4+T cells express a high-molecular weight isoform recognized by anti CD45RB monoclonal antibodies (CD45RA in humans), the reciprocal subsets on activated lymphocytes are identified by the loss of CD45RB monoclonal antibodies staining (Bell et al., 1998Go). Lymphocyte O-linked (Galß1,3GalNAc) glycans, reactive with PNA, are other markers which seem to be develop­mentally regulated (Wu et al., 1997Go), and have also been implicated in T-cell proliferation and differentiation (Barclay et al., 1987Go). PNA receptors are predominantly present in immature (cortical) thymocytes (CD4-CD8- and CD4+CD8+), and in mature CD4-CD8+ memory or activated lymphocytes (Galvan et al., 1998Go). The GalNAc specific lectin from Amaranthus leucocarpus recognizes murine medullary thymocytes (Lascurain et al., 1994Go) and human T-cells with the phenotype CD4+CD45Ra+CD27+, indicating that the lectin receptor is present specifically in naive or quiescent cell subpopulations (Lascurain et al., 1997Go). In this work we confirmed that the main proportion (83%) of the murine thymocytes recognized by ALL are CD4+CD8-; furthermore, we identified them as CD45RB+, indicating that they corres­pond to a naive cell subset of thymocytes, which could be destined for export to the peripheral lymphoid tissues (Sprent, 1989Go).

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, 1986Go; Piller et al., 1988Go).

By means of lectins with similar specificity to ALL, several authors characterized different leukocyte antigens; PNA recognizes a major glycoprotein of 170–180 kDa and minor bands of 110–120 kDa (De Maio et al., 1986Go); the lectin from Salvia sclarea interacts specifically with a 125 kDa glyco­protein that corresponds to leukosialin (CD43 or sialophorin) (Piller et al., 1988Go). Mucin-like structures have been identified in other leukocyte antigens (Shimizu and Shaw, 1993Go), 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, 1986Go; Shelley et al., 1989Go), 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., 1998Go), Fas-associated death domain protein (Fernandes-Alnemri et al., 1996Go), and transforming growth factor-ß type II receptor (Suzuki et al., 1994Go).

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., 1998Go), 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.


    Materials and methods
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 References
 
Reagents
Amaranthus leucocarpus seeds were obtained in Tulyehualco, Mexico, and the lectin was purified by affinity chromatography as described by Zenteno and Ochoa (1988)Go. The A.leucocarpus lectin (ALL) was labeled with the N-hydroxysuccinimide ester of biotin from Pierce Chem. Co. (Rockford, IL) at a label/protein ratio of 2:1 (Savage et al., 1992Go). Electrophoresis and blotting reagents were obtained from Bio-Rad Lab. Inc. (Richmond, CA, USA). Cell culture media, biotin, avidin monomeric–agarose, avidin–peroxidase, bovine serum albumin fraction V, sugars, and chemical reagents were from Sigma Chemical Co. (St. Louis, MO). Phycoerythrin (PE)-labeled antibodies against murine lymphocyte markers: CD2, CD3, CD4, and CD43 isoforms, as well as FITC-labeled anti-CD8, and anti-CD45RB, as well as rat anti-mouse antibodies conjugated with horseradish peroxidase were obtained from Pharmingen (New York, NY). Trypsine, sequence grade, was obtained in Promega (Orsay, France).

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 Dulbecco’s modified Eagle’s 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, 1983Go).

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, 1980Go). 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, 1986Go); 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 0–1 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)Go 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)Go, using bovine serum albumin as standard. Carbohydrate concentration was determined by the method of Dubois et al. (1956)Go, 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., 1972Go).

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)Go, 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 SDS–PAGE 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 SDS–PAGE 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., 1995Go).

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% SDS–polyacrylamide 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.


    Acknowledgments
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 References
 
Thanks are due to Marie-Christine Slomianny (USTL) for the MALDI-TOF analysis. This work was supported in part by CONACyT (27609 M), PAED (202317) and DGAPA (PAPIIT-IN224598) UNAM, and by Program ECOS Mexico-France (M97B05).


    Footnotes
 
1 To whom correspondence should be addressed at: Laboratorio de Inmunologia, Departamento de Bioquimica, Facultad de Medicina UNAM, PO Box 70159, 04510 Mexico Back


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 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 References
 
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