Endogenous Lectins from Cultured Cells ISOLATION AND CHARACTERIZATION OF CARBOHYDRATE-BINDING PROTEINS FROM 3T3 FIBROBLASTS*

Extracts of cultured 3T3 fibroblasts, obtained by homogenization and Triton X- 100 solubilization, were fractionated on Sepharose columns covalently deriva- tized with asialofetuin. Three distinct carbohydrate-binding proteins (CBPs) were purified from the mate- rial bound to the affinity column: CBP35 (Mr = 35,000), CBP16 (Mr = 16,000), and CBP13.5 (Mr = 13,500). These CBPs were similar in several key properties. (a) They showed agglutination activity when assayed with rabbit erythrocytes; (b) they all appear to specifically recognize galactose-containing glyco- conjugates; (c) they have low isoelectric points, PI 4.5-4.7; (d) their binding activities are rapidly lost in the absence of p-mercaptoethanol; (e) the CBPs do not interact with each other, and the fractionated proteins can bind to asialofetuin independent of associated polypeptides; and ( f ) none of the proteins tend to self- associate to form oligomers of identical subunits. Comparisons of these and other properties of the CBPs suggest that CBPl6 and CBP13.5 may be the murine counterparts of lactose-specific lectins previously identified in electric eel and in several bovine and avian tissues. In contrast, it appears that CBP35 represents a newly identified protein capable of binding to galactose-containing carbohydrates. Tris-HC1, 0.1 M NaCl, pH 7.4, and finally with water. The pellet was dissolved in 100 pl of buffer for polyacrylamide gel electrophoresis and boiled for 2 min prior to electrophoresis on 10% polyacrylamide gels.

The purification of CBPs,' including lectins and enzymes such as glycosyltransferases and glycosidases, has made much use of the powerful technique of affinity chromatography. Because of the similarity of saccharide structures found on various serum glycoproteins such as fetuin and those found on the cell surface (1-3), CBPs can potentially recognize similar monosaccharide units, oligosaccharide structures, or the entire carbohydrate complex on glycoproteins and on cell surface heterosaccharides. This suggests that affinity columns containing Sepharose covalently coupled to a glycoprotein such as fetuin might be used for the isolation of CBPs. Indeed, * This work was supported by Grant PCM-8011736 from the National Science Foundation, Grant BC-277A from the American Cancer Society, and Grant GM-27203 from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
$Supported by a fellowship from the Michigan State University College of Osteopathic Medicine.
$Supported by Faculty Research Award FRA-221 from the American Cancer Society.
We have undertaken a study of CBPs from an established tissue culture cell line, Swiss 3T3 fibroblasts. This cell line has well defined growth and morphological characteristics (9,10). Because it is thought to be derived originally from mouse embryo fibroblasts, 3T3 cells presumably represent cells of a rather ubiquitous distribution. In the present study, we report the purification and characterization of three distinct CBPs, all of which were isolated on the basis of their binding to ASF-Sepharose and subsequent elution with the disaccharide lactose. The CBPs exhibited agglutination activity for rabbit erythrocytes and therefore are probably fibroblast lectins.

EXPERIMENTAL PROCEDURES
Materials-Swiss 3T3 cells were obtained from American Type Culture Collection (CCL92). Dulbecco's modified Eagle's medium was from K. C . Biologicals, calf serum from M. A. Bioproducts, and fetuin from Gibco. All carbohydrates, cyanogen bromide, and phenylmethylsulfonyl fluoride were products of Sigma; Aquacide I11 of Calbiochem-Behring; and Sepharose 4B and Sephadex G-150 of Pharmacia Fine Chemicals. [3SS]Methionine (1012 Ci/mmol) was bought from New England Nuclear. Ampholine was purchased from LKB.
Culture and Radiolabeling of 3T3 Cells-Maintenance of Swiss 3T3 cells has been described elsewhere (11). Swiss 3T3 cells were grown to confluent monolayers (4-5 X lo4 cells/cm2). The medium was replaced with fresh growth medium for 24 h. This medium was removed and the cells were cultured in serum-free Dulbecco's modified Eagle's medium (10 m1/150 cm2 of growth area) containing 3 pg/ ml of unlabeled methionine (one-tenth of the concentration normally found in Dulbecco's modified Eagle's medium) and 20 pCi/ml of [35S] methionine (12). After 24 h, the medium was removed, and the cells were washed prior to the extraction and isolation of the proteins.
Preparation of Asialofetuin-Sepharose-Fetuin was desialylated as described by de Waard et al. (13) and coupled to Sepharose 4B by the method of Cuatrecasas (14). Fetuin (500 mg) dissolved in 25 ml of H20, pH 2.0, was heated at 80 "C for 1 h. The solution was then cooled to 25 "C, neutralized with NaOH, and dialyzed against 0.2 M NaHC03, pH 7.9. The ASF was coupled to 150 ml of CNBr (20 g)activated Sepharose 4B in a combined volume of 300 ml of 0.2 M NaHC03, pH 7.9. After 24 h at 4 "C, 150 ml of 2 M ethanolamine, pH 8.0, were added for an additional 24 h. The resin was washed extensively with 1 M NaCl and then washed with Buffer A (see below).
Routinely, greater than 80% of the ASF was coupled as determined by the difference in absorbance at 280 nm of the ASF solution before and after the coupling reaction.
Isolation of Asialofetuin-binding Proteins-The purification of from 3T3 Cells fonyl fluoride. The cells in each flask were scraped with a rubber policeman into 2 ml of Buffer B. The pooled cellular material was homogenized in a 2-ml Potter homogenizer (102-152-pm clearance) at five strokes/2 ml. Insoluble material was pelleted by centrifugation at 3000 X g for 15 min, and then the supernatant was cooled to 4 "C. All previous operations were performed at 25 "C and subsequent steps a t 4 "C. The supernatant was applied to an ASF-Sepharose column (1.4 X 15 cm), and the column was washed extensively with Buffer B containing 2 mM @-mercaptoethanol. To remove detergent, the column was washed with 2-4 column volumes of Buffer C. Protein bound on the ASF-Sepharose column was eluted with a 0-0.15 M lactose gradient (100-ml total volume). Aliquots from the column effluent were assayed for radioactivity due to [35S]methionine-labeledproteins using scintillation counting (12).
The material eluted by lactose was pooled and dialyzed against Buffer C in tubing impermeable to molecules of molecular weight greater than 3500. The dialysis tubing also contained ASF-Sepharose. The dialyzed contents were poured into a column, and the resin was allowed to settle before washing with Buffer C. The bound proteins were eluted with a lactose gradient as described above.
The material eluted with lactose from the second affinity column was concentrated by reverse dialysis against Aquacide 111 to a final volume of 1-2 ml. This concentrate was chromatographed on a column (1 X 150 cm) of Sephadex G-150. Fractions from the Sephadex G-150 column were pooled and tested for their ability to rebind to another ASF-Sepharose column.
Gel Electrophoretic Characterization of CBPs-Polyacrylamide gel electrophoresis in SDS was performed according to the procedure of Laemmli (15) on a 5-16% gradient slab gel (1 mm X 9 cm) (0.21-0.67% bisacrylamide) with a 1-cm long 4% stacking gel. Samples were prepared by dialysis against water followed by lyophilization. They were dissolved in 1% SDS, 4% 0-mercaptoethanol and boiled for 1 min. After electrophoresis the gels were fixed for 30 min in 10% trichloroacetic acid and stained with Coomassie brilliant blue. After destaining the gel was subjected to fluorographic treatment as described by Bonner and Laskey (16), using Kodak X-Omat AR (XAR-5) film.
Two-dimensional gel electrophoretic analysis was performed according to the method of O'Farrell (17). Samples were first subjected to isoelectric focusing in tube gels (1 mm X 10 cm) containing pH 3-10 Ampholine. The second dimension was electrophoresed on 5 1 6 % polyacrylamide slabs as described above.
Assays of Agglutination and Enzymatic Actiuities-Fresh rabbit erythrocytes were isolated following the method of Lis and Sharon (18), and trypsin-treated, glutaraldehyde-fixed rabbit erythrocytes were prepared by the method of Nowak et al. (19). The cells were used as a 4% stock suspension in 0.9% NaCl containing 0.3% bovine serum albumin, pH 7.4. Hemagglutination assays were carried out in microtiter V-plates; each well contained 25 p1 of erythrocyte suspension and 25 p1 of the test sample. To study the effects of saccharides on hemagglutination, 10 pl of a stock solution of saccharide in 0.9% NaCl were added control wells received 10 pl of 0.9% NaCI. In addition, the effects of the various saccharides on the erythrocytes were tested in the ahsence of any agglutinin sample. All agglutination assays were scored after 1 h at room temperature. @-Galactosidase activity was determined by the method of Bishop and Desnick (20). To 150 p1 of 1.5 mM 4-methylumbelliferyl-P-Dgalactopyranoside (Pierce Chemical Co.) in 0.03 M citrate, 0.05 M phosphate, pH 4.6, were added 50 pl of the test sample. After incubation for 1 h at 37 "C, the reaction was terminated by the addition of 2.4 ml of 0.1 M ethylenediamine. Fluorescence was monitored on a Perkin-Elmer 650-40 fluorometer using excitation and emission wavelengths of 360 and 440 nm, respectively. 0-Galactosidase activity was also determined at neutral pH using 1.5 mM 4-methylumbelliferyl-8-D-galactopyanoside in Buffer B.
Sialyltransferase activity was determined by the method described by Bosmann (21) using ASF as a potential acceptor. To a solution of 10 mM MgCl,, 10 mM MnCl,, 400 pg of ASF, and 8.6 X lo4 dpm of cytidine 5'-monophospho-N-acety1[4,5,6,7-8,9-'4C]neuraminic acid (Amersham Corp., 247 mCi/mmol) in 100 pl of Buffer B were added 50 pl of test sample. After 4 h at 37 "C, an aliquot was removed and 5 volumes of cold 1% phosphotungstic acid in 0.5 N HCI were added. The mixture was centrifuged, and the precipitate was washed twice with 1% phosphotungstic acid in 0.5 N HC1, resuspended in 0.5 ml of H20, and neutralized with 1 N NaOH. The radioactivity was then determined by scintillation counting. Alternatively, the reaction mixture was chromatographed on columns (100 X 1.5 cm) of Sephadex G-25 and the effluent fractions were monitored for radioactivity. The test sample has also been assayed for transferase activity in the presence of 2 mM unlabeled CMP/sialic acid.
Preparation of Antisera and Immunoprecipitation-Antisera directed against CBP35 were raised in New Zealand White female rabbits. CBP35 isolated from 17 flasks (150 cm2 of confluent 3T3 fibroblasts) was mixed in 200 pl of complete Freund's adjuvant and injected near the popliteal lymph node (100 pl/node) of an etherized rabbit. After 10 days, the rabbit was injected at the same site with CBP35 (isolated from 10 flasks of confluent 3T3 cells) which was suspended in incomplete Freund's adjuvant. The rabbit was first bled 10 days after the second immunization. Subsequent hleedings were also made 10 days after boosting the rabbit. Antiserum against CLL I was prepared by injecting CLL I ( M , = 16,000) isolated from female adult chicken liver into a rabbit (22). It was a gift of Dr. Steven Ullrich (Michigan Molecular Institute, Midland, MI).
Material used for the immunoprecipitation was [35S]methioninelabeled 3T3 cell extract partially purified by affinity chromatography over one ASF-Sepharose column. The material was concentrated by reverse dialysis against Aquacide 111 to a volume of approximately 2 ml. The concentrated material was split into four aliquots, 450 pl/ tube, placed on ice for 1 h, after which it was centrifuged for 15 min a t 12,000 X g. The supernatants were transferred to new tubes and 5 pl of antisera were added. After incubation at 37 "C for 1 h, they were incubated at 4 "C for 8-12 h. Then, 150 p1 of goat anti-rabbit IgG serum (Gibco) were added to each sample, and they were placed at 4 "C for an additional 14 h.
The precipitates were pelleted by centrfugation at 10,000 X g for 15 min. The supernatant was discarded, and the pellet was washed twice in 0.05 M Tris-HCI, 1.2 M KC], 1% (v/v) Triton X-100, pH 7.4, followed by two washings in 0.05 M Tris-HC1, 0.1 M NaCl, pH 7.4, and finally with water. The pellet was dissolved in 100 pl of buffer for polyacrylamide gel electrophoresis and boiled for 2 min prior to electrophoresis on 10% polyacrylamide gels.

RESULTS
Asialofetuin-binding Proteins from 3T3 Cells"3T3 fibroblasts were cultured in the presence of [35S]methionine to label the cellular proteins. After washing, confluent monolayers of these labeled cells were extracted with Triton X-100 and fractionated by affinity chromatography on a column of ASF-Sepharose (Fig. 1). The majority of the radioactive material was not bound by the column (Fig. l, Component A). After extensive washing in buffer containing Triton X-100, the column was further developed with detergent-free buffer ( Fig. 1, position of arrow I). Finally, the column was eluted with a linear gradient of lactose (Fig. 1, position of arrow Z), which resulted in the appearance of a peak of radioactivity ( Fig. 1, Component C). The radioactivity in Component C ( Fig. 1) accounted for <0.01% of the total radioactivity applied to the affinity column.
Polyacrylamide gel electrophoretic analysis in SDS was carried out on Component C (Fig. l), as well as on '"S-labeled material from pooled fractions immediately before (Fig. 1,  Component B ) and immediately after (Fig. 1, Component D ) the radioactive peak. Component B (Fig. 1) yielded a heterogeneous mixture of polypeptides on SDS-gel analysis (Fig. 2,  lane b). In contrast, Component C ( Fig. 1) yielded three predominant bands, corresponding to M , = 35,000, 16,000, and 13,500 (Fig. 2, lane c ) . Several other bands were noticeable; they corresponded to M , = 30,000, 20,000, 11,000, and <10,000. Finally, Component D (Fig. l), which consisted of material eluted as a shoulder of the main radioactive peak, yielded a gel pattern that contained at least four prominent bands ( M , = 100,000, 36,000, 16,000, and 11,000), as well as many minor contaminants.
Component C (Fig. 1) can be further purified by another cycle of affinity chromatography. The pooled material was dialyzed to remove the lactose. Two important requirements were noted concerning the dialysis. ( a ) The presence of 0mercaptoethanol (2 mM) preserved the ASF-binding capacity of the proteins; in its absence, the binding activity of the preparation was completely lost. (b) The inclusion of the affinity matrix (ASF-Sepharose) in the dialysis bag prevented the nonspecific absorption of '"S-labeled proteins to the tubing and therefore increased the recovery of material during the dialysis procedure. After dialysis, the contents of the bag were packed directly into a column and washed. Approximately 20% of the dialyzed material did not bind to ASF-Sepharose; the bound material was eluted with lactose at a 220.  position similar to that found in the first affinity column (20 mM lactose).

Molecular Weights and Isoelectric Points of Asialofetuinbinding
Proteins-Polyacrylamide gel electrophoretic analysis in SDS of the material purified by two cycles of affinity chromatography on ASF-Sepharose yielded three polypeptide bands, with M , = 35,000, 16,000, and 13,500 (Fig. 2, lane f ) .
The material corresponding to these three bands will be referred to hereafter as CBP35, CBP16, and CBP13.5. Densitometric tracing of the fluorogram (Fig. 2, lane f ) showed that these three bands accounted for >99.9% of the total radioactivity and that the relative proportions of the individual bands were 2.9 (CBP35), 1.1 (CBPlG), and 1 (CBP13.5). Similar results were obtained both in the presence and absence of @-mercaptoethanol during the electrophoresis.
In some preparations, the material corresponding to highly purified CBPs (after two cycles of affinity chromatography) yielded a fourth band on polyacrylamide gels (Fig. 2, lane g). It accounted for no more than 2-3% of the total radioactivity on the gel. The molecular weight of this fourth band was estimated to be 34,000. Although the origin of this band is not known at present, this polypeptide also has the capacity to bind to ASF.
In order to determine the isoelectric properties of the polypeptides, a sample containing only CBP35, CBP16, and CBP13.5 was subjected to two-dimensional gel electrophoretic analysis (Fig. 3). The results showed that CBP16 and CBP13.5 had similar PI values of approximately 4.5. CBP35, which corresponded to a single band on one-dimensional electrophoresis, yielded two spots that had the same molecular weight ( M ' = 35,000) but different isoelectric points (PI 4.5 and 4.7) (Fig. 3). These results indicate that the ASF-Sepharose column could be used to isolate a minimum of three, and possibly four, polypeptides from 3T3 cells.
Effect of Saccharide Ligands and EDTA on Asialofetuinbinding Proteins-In order to probe the sugar-binding specificity of the ASF-binding proteins, Triton X-100 extracts of 3T3 cells were chromatographed on columns of ASF-Sepha-10660 Carbohydrate-binding Proteins from 3T3 Cells rose. Various saccharides were tested for their capacity to elute the bound radioactive polypeptides. When the column was developed sequentially with mannose (Fig. 4a, position of  arrow 3 , sucrose (position of arrow 3), and lactose (position of arrow 4), a prominent radioactive peak was observed only upon the addition of lactose (Fig. 413). Polacrylamide gel analysis in SDS of Components A, B, and C (Fig. 4a) showed that CBP35, CBP16, and CBP13.5 all were eluted with lactose (Fig. 5, lanes a-c).
When the ASF-Sepharose column was developed sequentially with N-acetyl-D-glucosamine (Fig. 4b, position of arrow  2), galactose (position of arrow 3), and lactose (position of arrow 4 ) , no radioactive material was eluted with the first monosaccharide (Fig. 4b). A peak of radioactivity was eluted, however, upon the addition of galactose (Fig. 46, Component  B ) . Polyacrylamide gel analysis in SDS of this material yielded two predominant polypeptides, corresponding to CBP35 and CBP13.5 (Fig. 5, lane e). When lactose was used to develop the column after galactose, some radioactivity was eluted in a rather ill defined peak (Fig. 4b, Component C). This material yielded CBPl6 upon SDS-gel electrophoresis (Fig. 5, lane f ) .
These results indicate that the ASF-binding polypeptides are CBPs.
The question arose whether any of the CBPs had an intrinsic requirement for Ca'+ ion in order to bind the saccharide. To test this, the CBPs bound on ASF-Sepharose were eluted with Cay+-free buffer containing 10 mM EDTA. No distinct , " " " " _"   (Fig. 4). Fractions from Fig. 4a:  lane a, Component A; lane b, Component B; lane c, Component C. Fractions from Fig. 46: lane d, Component A; lane e, Component B; lane f, Component C. Approximately 4000 cpms were applied to each lane, and the fluorograms were exposed for 30 days (lanes a-c) or 45  days (lanes d-f).
peak of radioactivity was observed. Gel analysis in SDS of the pooled fractions eluted with EDTA revealed trace amounts of CBP35, CBP16, and CBP13.5. There was no apparent enrichment of any one of the polypeptides relative to the other two.
In contrast, the addition of lactose to the ASF-Sepharose column after EDTA yielded a substantial peak of radioactivity. All three of the ASF-binding polypeptides (CBP35, CBP16, and CBP13.5) were found in the fractions eluted with lactose. It appears, therefore, that none of the CBPs require Ca2+ ions for saccharide binding. This conclusion is further corroborated by experiments in which the same three CBPs were isolated from the ASF-Sepharose column when the extraction and affinity chromatography were carried out in calcium-free buffer (Buffer A was replaced by 75 mM NaCl, 2 mM EGTA, 2 mM NaN3, and 75 mM Tris, pH 7.0).
Fractionation of the Carbohydrate-binding Proteins-Gel filtration of a mixture containing CBP35, CBP16, and CBP13.5 on Sephadex G-150 further fractionated them into two new components (Fig. 6). Component A (Fig. 6) chromatographed to a region corresponding to M, = 30,000-35,000.
Upon gel electrophoresis in SDS, it yielded only CBP35 (Fig.  7, lanes b and d ) . (In the sample used for Fig. 7, the gel also shows a minor band corresponding to a molecular weight of about 34,000; this represents the fourth band of the CBPs that occurs in some preparations.) Gel electrophoresis in SDS of Component B (Fig. 6) showed that it consisted of only CBP16 and CBP13.5 (Fig. 7, lanes c and e). No CBP35 was observed in these fractions. Similar results were obtained both in the presence as well as in the absence of lactose (Figs.  6 and 7).
These results indicate that CBP35 does not interact with either CBP16 or CBP13.5. In addition, the position of migration of CBP35 (Fig. 6, Component A ) suggests that the molecular weight of the protein in the absence of denaturants is 35,000, and therefore the polypeptide does not self-associate into dimers or oligomers. Similarly, the chromatographic position of Component B (Fig. 6) is consistent with polypeptides of M, = 13,000-16,000. Moreover, we have achieved a similar fractionation using columns of Sephadex G-50. Under this condition, the chromatographic position of CBP16 and CBP13.5 was well resolved from that of chymotrypsinogen (M, = 25,000). Therefore, it appears that CBP16 and CBP13.5 do not bind to each other in nondenaturing solvents.   G-150 (Fig. 6). Lane a is an aliquot of the sample which was applied to the column. Intrinsic Binding Properties of the Carbohydrate-binding Proteins-Component A (Fig. 6) was applied to an ASF-Sepharose column. More than 95% of the radioactivity applied was bound to the affinity column and could be eluted with lactose. Polyacrylamide gel electrophoresis in SDS of the recovered material showed that it was highly purified CBP35. This suggests that CBP35 can bind to carbohydrates independently of CBP16 and CBP13.5. Moreover, isolated CBP35 can agglutinate rabbit erythrocytes as well as rabbit erythrocytes previously treated with trypsin followed by glutaraldehyde fixation (Table I). This agglutination was inhibited by lactose (0.05 MI. Component B (Fig. 6), which consisted of CBP16 and CBP13.5, also exhibited lactose-inhibitable agglutination activity (Table I). Component B (Fig. 6) can be bound to ASF-Sepharose columns and eluted with specific saccharides. Elution with galactose yielded a fraction containing only CBP13.5. Subsequent elution with lactose yielded a fraction containing CBP16, although this protein can also be detected at the latter part of the galactose elution (0.15 M galactose). These results corroborate the previous demonstration that galactose can elute CBP13.5 and lactose can elute the bulk of the CBP16 from ASF affinity columns (Fig. 4b). Together, they suggest that both CBP16 and CBP13.5 have intrinsic carbohydrate-binding capacities, independent of other associated polypeptides.
We have also tested the various fractions containing the CBPs for enzymatic activities such as glycosidases and transferases. Using 4-methylumbelliferyl-~-~-galactopyranoside as a substrate, we found no &galactosidase activity associated with the CBPs at pH 4.6 and at 7.2 (Table I). Moreover, all of the &galactosidase activity observed in the original cell extract could be accounted for in the material not bound by the first ASF-Sepharose column (Fig. 1, Component A). Similarly, we found no transferase activity associated with the CBP fractions as assayed with I4C-labeled CMP/sialic acid and ASF. This conclusion is based on experiments which showed that the radioactivity precipitated along with ASF by phosphotungstic acid was identical when the transferase assay was carried out in the presence and absence of the CBPs. Moreover, analysis of the assay reaction mixture by chromatography on Sephadex G-25 showed that no radioactivity migrated in the void volume fractions, at a position corresponding to ASF.
Immunoprecipitation of the Carbohydrate-binding Proteins-The availability of two antisera, one directed against CBP35 (anti-CBP35) and the other directed against CLL I N T C) CBPs 35.16,and 13.5 (after two cycles of + --
(anti-CLL I), provided the necessary reagents to study the structural relationships between CBP35, CBPl6, and CBP13.5, as well as to test for relatedness to lectins characterized in the chicken intestine and muscle systems. Component C (Fig. I), which contained a mixture of CBP35, CBP16, and CBP13.5, was subjected to immunoprecipitation by anti-CBP35 and anti-CLL I. Analysis of the immunoprecipitates by gel electrophoresis showed that anti-CBP35 reacted only with CBP35 but not with CBP16 and CBP13.5 (Fig. 8, lane   b). In contrast, anti-CLL I precipitated only CBP16 and no other CBP from the mixture (Fig. 8, lane c). These results suggest that CBP35 is not structurally related and therefore is most probably not a higher molecular weight precursor of CBP16. In addition, the immune reactivity of CBP16 with anti-CLL I indicates that this CBP is probably a murine fibroblast counterpart of the lactose-specific lectins described in a number of other systems. DISCUSSION The results obtained in the present study indicate that we have purified, from the 3T3 fibroblast system, three distinct  5 (M, = 13,500). These three proteins are similar in several key properties. First, they all appear to recognize specifically galactose-containing glycoconjugates. In the present paper, we have isolated them on the basis of their binding to columns containing the asialoglycoprotein ASF, and we have demonstrated differences between them on the basis of their elution from the affinity column using lactose or galactose. We have also obtained evidence that these proteins will bind polyacrylamide beads derivatized with disaccharide ligands of defined structure (DGal@(l-t)PDGlcNAc) but not to beads containing only the monosaccharide /3DGal (23). Moreover, fractions containing CBP35 or CBP16 and CBP13.5 exhibited agglutination activity when assayed with rabbit erythrocytes. This agglutination can be inhibited by lactose. These binding and agglutinating results demonstrate unequivocally that the isolated proteins are carbohydratebinding proteins.
Second, these CBPs have low isoelectric points (PI 4.5-4.7), indicating that they are most probably acidic proteins. In our two-dimensional gel electrophoretic analysis, CBP35 was actually resolved into two different spots with PI values of 4.5 and 4.7. The relationship of these two polypeptides, of the same molecular weight but of different isoelectric points, has not been determined.
Third, the binding activity of all three of the CBPs was rapidly lost in the absence of /3-mercaptoethanol. This suggests that functional groups, most likely free sulfhydryl or tryptophan residues (24), may be sensitive to air oxidation. Fourth, CBP35, CBP16, and CBP13.5 do not appear to require Ca2+ for activity.
Finally, gel filtration studies in nondenaturing solvents indicate that the CBPs do not interact with each other, both in the presence and absence of lactose. The chromatographic data also suggest that none of the three polypeptides appear to self-associate to form oligomers of identical subunits. Because these column separation experiments were carried out using minute amounts of radiolabeled proteins, however, the concentrations of CBPs used in our column experiments may be below the threshold required for aggregation. It is also possible that the reducing agent that favors carbohydratebinding activity blocks linkage by disulfide bonds. Therefore, association might occur under other conditions. In any case, it should be emphasized that each of the fractionated proteins can bind to ASF, independent of associated polypeptides.
Comparisons of the polypeptide molecular weights, the isoelectric points, agglutination activity, and the carbohydrate-binding specificity of CBP16 and CBP13.5 with the lactose-specific lectins isolated from electric eel organ (24), calf heart (7) and lung (13), and chicken intestine (25) and muscle (19,26) suggest that they may be related proteins. This conclusion is supported by the observation that antibodies raised against CLL I (M, = 16,000) immunoprecipitated only CBP16 out of a mixture containing all three CBPs. In addition, these proteins all share the similar properties of being highly sensitive to air oxidation and of being calciumindependent. CBP16 and CBP13.5 differ from these lactosespecific lectins in one major respect. Whereas many of the previously identified lectins self-associated to form dimers and oligomers, CBP16 and CBP13.5 remain in monomeric form. This is similar to the behavior of CLL 11, which also remains as a monomer (8). If these proteins do indeed turn out to be analogous counterparts of each other in different species (27,28), then the question concerning their postulated tissue-specific functions(s) must be raised. For example, if the developmentally regulated lactose-extractable lectins from myoblasts function as mediators of cell-cell fusion in the formation of myotubes (29,301, the specificity of these events must have more strict constraints other then carbohydrate recognition. Perhaps they have multiple and more general functions, such as organizing complementary glycoconjugates, as was inferred from studies of CLL I in the development of chicken pectoral muscle in primary cultures (31).
To the best of our knowledge, a protein analogous to CBP35 has not been previously identified and isolated in other species or from other cell types. It does not appear that CBP35 is a higher molecular weight precursor to CBP16 and/or CBP13.5. Antibodies directed against CBP35 did not show cross-reactivity with either CBP16 or CBP13.5. Conversely, antibodies that recognized CBP16 failed to react with CBP35. Therefore, CBP35 most probably represents a new carbohydrate-binding protein, co-isolated with CBP16 and CBP13.5, which do have analogous counterparts. At present, it does not appear that CBP35 is the fibroblast counterpart of the galactose-specific receptor on hepatocytes, whose properties have been reported and reviewed by Ashwell and Harford (32), Stockert et al. (33), Kawasaki and Ashwell (34), Tanabe et al. (35), and Hudgin et al. (36.) The differences between the two proteins include ( a ) molecular weight of the polypeptide chain, ( b ) aggregation properties of the polypeptides, ( c ) effect of EDTA on binding of carbohydrates, and (d) sensitivity to air oxidation.
We have recently purified a carbohydrate-binding protein from mouse lung tissue using the same procedures described for the isolation of CBP35. The molecular weight of this protein was 35,000, as determined by SDS-polyacrylamide gel electrophoresis and Coomassie blue staining. When the mouse lung protein on the polyacrylamide gel was transferred onto nitrocellulose paper and then immunoblotted with anti-CBP35, a single radioactive band (M, = 35,000) was observed after autoradiography. These results suggest that we can isolate CBP35 in large amounts (microgram levels) from mouse lung. This in turn will allow us to carry out structural studies on the polypeptide, to study its cellular localization, and to search for its endogenous ligand in the cell.