Purification and characterization of a glycopeptide derived from phaseolus vulgaris leukoagglutinating phythohemagglutinin

386

Biochimica et Biophysica Acta, 579 (1979) 3 8 6 - - 3 9 5 © E l s e v i e r / N o r t h - H o l l a n d Biomedical Press

BBA 38240

PURIFICATION AND CHARACTERIZATION OF A GLYCOPEPTIDE DERIVED FROM P H A S E O L U S V U L G A R I S LEUKOAGGLUTINATING PHYTHOHEMAGGLUTININ

R O B E R T HSU *, J. B R U C E M I L L E R , S T A N L E Y Y A C H N I N and GLYN DAWSON

Departments o f Medicine, Pediatrics, and Biochemistry, The University o f Chicago Pritzker School o f Medicine, The Joseph P. Kennedy, Jr. Mental Retardation Research Center, and The Franklin McLean Memorial Research Institute, Chicago, IL 60637 (U.S.A.) (Received J a n u a r y 5th, 1979)

Key words: Phytohemagglutinin; Lectin; Glycoprotein; Oligosaccharide structure

Summary A glycopeptide obtained from tryptic digestion of citraconylated leukoagglutinating (L-subunit) phytohemagglutinin was isolated and purified. The composition of its ten amino acid and seven hexose residues, when compared to a previous analysis of the NH2-terminal amino acid sequence, indicated that the glycopeptide was derived from residues 11 through 20, with the oligosaccharide unit linked N-glycosidically to asparagine at the twelfth position. Structural studies involving a combination of a-mannosidase digestion, periodate oxidation, and permethylation, in conjunction with computerized mass-spectrometric and masschromatographic techniques, suggested a branched sequence of five mannose and two gtucosamine residues similar to that found in many mammalian glycoproteins.

Introduction The five phytohemagglutinin mitogenic proteins (PHAP) obtained from the red kidney bean, Phaseolus vulgaris, are isomeric, non-covalently bound tetrameric glycoproteins [1,2] which are made up of varying proportions of two different subunits, previously designated as L and R [3,4]. One of the five glycoproteins, L-PHAP, is homogeneous and is comprised of four identical L subAbbreviations: L-PHAP, leukoagglutinating phytohemagglutinin; H-PHAP, hemagglutinating phytohemagglutinin; GlcNAc, N-acetylglucosaraine; HexNAc, N-acetylhexosamine. * To whom reprint requests should be addressed.

387 units; it is a potent mitogen and leukoagglutinin with low hemagglutinating activity [ 1 ]. In contrast, the 4R tetramer shows little or no lymphocyte mitogenic activity, but is a potent hemagglutinin. The L and R subunits have identical molecular weights of approximately 34 000 but differ in their isoelectric point and have been obtained in homogeneous form by isoelectric focusing. Partial sequence analysis indicates that, although they differ in six of the first seven residues from the NH2-terminus, they are identical in positions 8 through 24 [4]. The amino acid compositions and tryptic peptide maps of the L and R subunits are, in fact, strikingly similar [5]. The twelfth residue in each subunit is a glycosylated asparagine, and the compositions of the carbohydrate moieties are nearly identical, consisting of two N-acetylglucosamine (GlcNAc) and from five to six mannose residues. Recently Lis and Sharon reported structural studies on the carbohydrate unit of soybean agglutinin, which has approximately nine mannose and two GlcNAc residues [6]. However, very little other information is available about the oligosaccharide units of sugarbinding lectins or, indeed, of many plant glycoproteins. We have therefore attempted the structural analysis of a highly purified glycopeptide obtained from L-PHAP (pure L subunit) which contained the C-12 asparaginyl-linked oligosaccharide unit. Materials and Methods

Materials. The isolation and purification of native tetrameric L- and H-PHAP from commercial Bactophytohemagglutinin P (Difco Laboratories, Detroit, Michigan; catalog No. 3110-59), and the isolation of homogeneous L- and Rsubunits by isoelectric focusing in 8 M urea of L- and H-PHAP, have been decribed previously [4]. Trypsin (code TRTPCK), lot 34M600, was obtained from Worthington Biochemical Corp., Freehold, NJ. ~-Mannosidase (EC 3.2.1.24) from jack-bean meal, and N-acetyl-~-glucosaminidase (EC 3.2.1.30) from beef kidney, were purchased from Boehringer Mannheim Co., Indianapolis, Indiana. Citraconic anhydride was obtained from Pierce Chemical Co., Rockford, IL, and ~-naphthol, from Eastman Kodak Co., Rochester, NY. The ~naphthol was further purified by sublimation before use. Sodium meta-periodate was purchased from J.T. Baker Co., Phillipsburgh, NJ. Sephadex and BioGel chromatography materials were the products of Pharmacia Fine Chemicals, Piscataway, NJ, and Bio-Rad Corp., Richmond, CA, respectively. Preparation of L-PHAP glycopeptide. Large peptide fragments were initially prepared from pure L-PHAP by reaction of the denatured protein with citraconic anhydride, a specific and easily removable reagent for blocking of the amino group of lysine residues [7]. The citraconylated L-PHAP (125 mg)was dissolved in 3 ml of 0.1 M Tris-HC1 buffer (pH 8.5) containing 8 M urea, and the resultant clear solution was further diluted with 9 ml of 0.1 M Tris-HC1 buffer (pH 8.5) containing 0.1% thiodiglycol. Digestion with 0.75% {w/w) Trypsin-TPCK was carried out for 24 h at room temperature, with a second addition of 0.75% trypsin at 4 h. The reaction mixture was acidified to pH 4 with acetic acid and left standing at 4°C overnight. By this last step, trypsin digestion was terminated and the citraconyl blocking groups were removed simultaneously. The entire digested material was applied to a Sephadex G-50 superfine column (2.5 X 190 cm). The glycopeptide {identified by the ~-

388 naphthol-H2SO4 m e t h o d [8] was eluted with 0.01 M NH4OH as a single, but rather broad peak behind the exclusion volume of the column. It was then applied to a Sephadex G-25 superfine column (2.2 X 194 cm) and eluted with 0.01 M NH4OH. The glycopeptide fraction obtained was lyophilized, dissolved in 5 ml of starting buffer (0.2 M pyridine/acetate, pH 3.15), and applied to a column (0.9 X 26.0 cm) of previously equilibrated SP-Sephadex C-25. The column was washed with 90 ml of starting buffer and eluted with a linear gradient of pyridine in water (250 ml of pyridine/acetate buffer (2.0 M pyridine) at pH 5.0, into an equal volume of starting buffer). Final purification of the glycopeptide was achieved by preparative descending paper chromatography in 1-butanol/acetic acid/water (4 : 1 : 5; v/v, upper layer) [9]. Analytical methods. Amino acid analysis of the glycopeptide was performed with a Durrum D-500 analyzer [ 10] following hydrolysis of the salt-free sample in redistilled, constantly boiling HC1 in vacuo at 110°C for 24 h. C o m p o n e n t sugars were identified and quantitated by gas-liquid chromatography, as described previously [11,12]. We assessed the purity of the glycopeptide by t w o dimensional chromatography and electrophoresis, as described previously

[151. Digestion with purified glycosidase. The glycopeptide (0.5 mg) was digested with jack-bean a-mannosidase (5 units) in 0.05 M sodium citrate buffer, pH 4.4 (0.2 ml), for 20 h at 37°C [13], after which the reaction was stopped by immersion in a boiling water bath for 2 min. The reaction mixture was centrifuged and the precipitate washed with 0.2 ml of water. After centrifugation, the combined soluble material was applied to a Bio-Gel P-2 (1.0 X 160 cm) column and eluted with distilled water to resolve liberated mannose from glycopeptide. Both fractions were analyzed for hexoses by gas-liquid chromatography [11]. Periodate oxidation. Periodate oxidation of the glycopeptide (200 ttg) was carried o u t in 0.015 M sodium metaperiodate as described previously [14]. Following the removal of excess borohydride with glacial acetic acid and methanol [14], the residue was dissolved in 1 N HC1 in dry methanol, hydrolyzed at 80°C for 16 h, and analyzed by gas-liquid chromatography of the trimethylsilylated methylglycosides [ 11 ]. Permethylation. Purified glycopeptide (0.5 mg) was exhaustively methylated b y iodomethane in the presence of methylsulfinylanion in dimethylsulfoxide [15]. Following subsequent acetolysis and hydrolysis, the mixture of partially methylated mannose and glucosamine was reduced to the alditol form, acetylated, and separated by gas-liquid chromatography on a column of 3% OV-210, as described previously [16]. Derivatives were identified with an LKB-9000 data-handling system [ 17 ]. Mass chromatography. Partially methylated alditol acetate mixtures were examined by mass chromatography [ 18,19,20], in which repetitive scanning was carried o u t every 5 s over the mass range role 15--400. In this way, it was possible to obtain normalized spectra showing the relative abundance of a particular ion at different points in the chromatograph. Furthermore, from the relative retention times and the mass spectra of each eluted methylated alditol acetate, in conjunction with standards and with values reported previously [21], each methylated neutral sugar could be identified unequivocally.

389 Results

Composition of the glycopeptide. The composition of the glycopeptide obtained from the L subunit of phytohemagglutinin is shown in Table I. The amino acid and amino sugar values are taken from the 6 N HC1 hydrolysate, with the data presented as mol of amino acid and sugar per mol of glycopeptide, assuming one mol of the isoleucine per mol of glycopeptide. The mannose value (4.9) relative to the GlcNAc value (2.1) was obtained from gas-liquid chromatography of the trimethylsilylated sugars [11,12]. The results of amino sugar analysis by the t w o procedures were in good agreement, and the ratio of mannose to amino sugar and isoleucine was calculated as 5.0 : 2.1 : 1.0. The glycopeptide contained a total of 10 amino acid and 7 hexose residues. Digestion with ~-mannosidase. Enzymatic degradation with the exo-glycosidase ~-mannosidase showed that four of five mannose residues were readily released, and that the remaining mannose residue was linked to the asparagine residue of the peptide backbone. Incubation of the Man(GlcNAc)2-Asn-glycopeptide with N-acetyl-~-D-glucosaminidase did not release any GlcNAc. Periodate oxidation. Periodate oxidation of the glycopeptide destroyed 4 of 5 mol of mannose, b u t none of the GlcNAc residues. This suggested either that one of the mannose residues in a linear sequence was substituted at C-3 or that there was a branch point (C-3 and C-6; C-2 and C-3; or C-3 and C-4) among the mannose residues. Permethylation studies. A gas chromatogram of the partially methylated alditol acetates obtained from the sugars of the L-subunit glycopeptide is shown in Fig. 1. There were three major hexose peaks and only one amino sugar peak. These were identified on the basis of massspectrometric analysis as 2,3,4,6-tetra-O-methyl,l,5-di-acetyl-mannitol (peak A); 3,4,6-tri-O-methyl-l,2, 5-tri-O-acetyl-mannitol (peak B); 2,4-di-O-methyl-l,3,5,6-tetra-O-acetyl-mannitol (peak C); and N-methyl-N-acetyl-3,6-di-O-methyl-4,5-di-O-acetyl-2-amino2-deoxy-glucitol (peak D), respectively. Under these conditions, the yield of tetra-O-methyl derivatives is only 60% of that of tri-O-methyl derivatives, and the data were corrected accordingly. The other apparent major peak was iden-

TABLE I CARBOHYDRATE AND AMINO ACID COMPOSITION OF GLYCOPEPTIDE Residues

Molar ratio *

Residues

Molar ratio *

Aspartic acid

2.00 0.91 0.21 1.97 0.04 0.12 0.18 1.00

Leucine Phenylalanine Histidine Lysine Arginine M a n n o s e * ** GlcNAc

1.84 1.03 0.03 0.06 0.99 4.95 2.11

Yield (%)

31

Threonine Serine Glutamic acid Proline

Glycine Alanine Isoleucine

(2) ** (1) (0) (2) (0) (0) (0) (1)

(2) (1) (0) (0) (1) (5) (2)

* T h e m o l a r r a t i o s are c a l c u l a t e d relative t o i s o l e u c i n e ( 1 . 0 0 ) . ** I n d i c a t e s t h e n e a r e s t i n t e g e r . *** M a n n o s e value w a s relative t o t h a t f o r G l c N A c , w h i c h w a s o b t a i n e d f r o m gas-liquid c h r o m a t o g r a p h y .

390

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tiffed as hydrocarbon impurities (plasticizers) (peak P), a common problem in mass-spectrometry. There were no other hexose-derived peaks higher than 10% of peak B. Five minor peaks corresponded to 2,4,6-tri-O-methyl-l,3,5-tri-O-acetyl-mannitol (peak E); 2,3,6-tri-O-methyl-1,4,5-tri-O-acetyl mannitol (peak F); 4,6-di-O-methyl-l,2,3,5-tetra-O-acetyl-mannitol (peak G); 3,6-di-O-methyl-l,2, 4,5-tetra-O-acetyl-mannitol (peak H); and 3,4-di-O-methyl-l,2,5,6-tetra-O-acetyl mannitol (peak I). These presumably represent some minor heterogeneity in the oligosaccharide moiety. The relative areas (molar ratios) of the major peaks A, B, C, and D are given in Table II. The data correspond to two terminal mannose residues, two C-2TABLE

II

AREA RATIOS OF PARTIALLY METHYLATED DEOXYALDITOL, ALDITOL, AND AMINODEOXYALDITOL ACETATES OBTAINED FROM A GLYCOPEPTIDE DERIVED FROM RED KIDNEY BEAN PHYTOHEMAGGLUTININ Peak

Compound

Relative area ratios

A

2 , 3 , 4 , 6 - t e t r a m e t h y l m a n n i t ol 3,4,6-trimethylm annitol 2,4-dimethylmannitol 2-deoxy-2(N-methyl-ace tamid o)-3,6-dimethylglucitol 2~4 ~6 - t r i m e t h y l m a n n i t o l 2, 3 , 6 - t r i m e t h y l m a n n i t o 1 4,6-dimethylmannitol 3 , 6 - d i m e t h y l m a n n i t ol 3,4-dimethy lmannitol

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intensities

substituted mannose residues, a single 3,6-disubstituted mannose residue, and a C-4-substituted GlcNAc residue. The technique used is not strictly quantitative because of variations in the volatility of the derivatives and because of the low level of recovery of N-acetylhexosamine derivatives. The major peaks (A, B, C, D) were further identified by mass chromatoggraphy, in which the whole chromatograph was scanned at a single ion intensity. Fig. 2 gives a typical example of such a scan. The fragments have been

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characterized previously [21]. Fragment role 101 is found in all three mannose derivatives. Fragment role 116 is seen only in peak D and is specific for a 4-substituted HexNAc. Fragment role 117 requires that C-2 be unsubstituted; it is a major fragment in peaks A and C. Fig. 3 provides confirmatory evidence of the contaminant peak, since role 149 is characteristic of plasticizers. Fragment m/e 145 is significant in both peaks A and B (this requires C-3, 4, and 6 of a hexose to be unsubstituted); m/e 129 (a major fragment derived from C-2-substituted hexoses) is significant in peak B. Mass chromatography. Additional confirmation of structural assignment is provided by the mass chromatograms in Fig. 4: m/e 189 (a major fragment from both C-2- and C-3,6-substituted mannose residues) is found in peaks B and C; role 161 (a major fragment in hexoses unsubstituted at C-3, -4, and -6) is found in peaks A and B, and role 158 (derived from C-4-substituted HexNAc) is a significant fragment in peak D [21]. Discussion

Phytohemagglutinin was the first lectin shown to be a mitogen [22]. In recent years, it has gained wide application in the study of l y m p h o c y t e transformation as a measure of immunologic reactivity and in the detection of surface changes occurring on human leukemic leukocytes [23--25]. Lectins which bind mannose and N-acetylglucosamine residues such as those derived from jack-bean meal (concanavalin A), wheat germ agglutinin, and the garden pea agglutinin are devoid of covalently b o u n d carbohydrate; other lectins, such as PHAP, soybean agglutinin, and lima bean agglutinin, which bind other sugars as galactose, are known to be glycoproteins [26,27]. We have characterized a glycopeptide containing the twelfth amino acid resi-

393

due (asparagine) from the N-terminal end of L-PHAP and shown it to contain ten amino acid and seven sugar residues. We concluded that the glycopeptide was derived from residues 11 through 20 on the basis of the composition of the amino acids and hexoses when compared with the previously reported amino acid sequence of the NH2 terminus of L-PHAP and knowledge that position twelve is an asparagine residue linked N-glycosidically to an oligosaccharide unit composed of t w o N-acetyl glucosamine and 5--6 mannose residues [4,5]. Trypsin cleavage at positions Arg 10 and Arg 20 of the NH2 terminus of the L subunit would result in this glycopeptide. We have previously found that the carbohydrate compositions of both L and R subunits, as determined by gas-liquid chromatography of the derived trimethylsilyl methylglycosides, are nearly identical, with 10--11 mannose residues, 4 glucosamine residues, and one fucose residue per subunit (Miller and Dawson, unpublished results). This is in general agreement with the results of R/is~inen et al. [ 28], which indicated that the only sugar residues in crystalline kidney bean leukoagglutinin (L-PHAP) were mannose (14 residues per subunit)and N-acetylglucosamine (4 residues per subunit). This total carbohydrate composition, together with the present results for the oligosaccharide at the twelfth position, indicate that there must be two oligosaccharide units associated with the L subunit. We are presently in the process of isolating this second oligosaccharide, which must contain L-fucose in addition to mannose and glucosamine (Miller and Dawson, unpublished data). The sequence of monosaccharides was based on the results of sequential degradation by highly purified exo-a-mannosidase. Since only four of five mannose residues were eliminated in the process, it is evident that the other mannose residue is fl-linked [13,29]. The results obtained from periodate oxidation and sodium borohydride reduction studies were consistent with those from enzymatic degradation and further suggested either the presence of only a single branch point among the mannose residues, or a linear structure with substitution of one of the residues at C-3. The existence of a single mannose branch point was confirmed by permethylation studies [17,21]. Three major partially methylated and acetylated mannitol peaks were obtained following permethylation and acetylation of the glycopeptide. These corresponded quantitatively to two terminal mannose residues, two C-2-substituted mannose residues, and one C-3,6-disubstituted mannose residue. The only peak obtained for partially methylated and acetylated N-acetyl-glucosaminitol indicated a glycosidic linkage at C-4. The simplest structure suggested b y these data is therefore Man2(a, 1 -+ 2)Man(a, 1 -+ 6)\Man(/~ '

1

4)GlcNAcGlcNAc-Asn

Man4(a, 1 -+ 2)Man(a, 1 -+ 3) /

Such a structure has been found previously in a number of mammalian glycoproteins [29,30], most notably the immunoglobulins. During the course of these studies, Lis and Sharon reported a basically similar structure for the core region of the carbohydrate unit of soybean aggiutinin [6]. The Man3GlcNAc branched core structure appears to be c o m m o n to both plant and animal kingdoms and indicates a remarkable evolutionary conservation of sugar sequences.

394

Despite these conclusions, the present studies do not eliminate the possibility of an asymmetric structure for the glycopeptide, such as: Man(or, 1 -* 2 ) M a n ( a , 1 -~ 2 ) M a n ( a , 1 --- 3 ) M a n ( ~ , 1 ÷ 4 ) G l c N A c ( ~ , 1 ~ , l ) G l c N A c /

Man(0G 1 ÷ 6)

Resolution of this and other structural possibilities will require more material than is currently available, since proof of the above would require the demonstration of derived Man(a, 1 -~ 2)-linked mannobiose, as shown by Lis and Sharon [6]. The homogeneity of the oligosaccharide units of the L-PHAP glycopeptide in terms of mannose and GlcNAc residues is similar to that observed for ribonuclease B [30,31], but in contrast to the heterogeneity of other mannose rich glycoproteins such as ovalbumin [3,29]. The carbohydrate units of many glycoproteins of mammalian origin are heterogeneous (for example, Dawson and Clamp [14]; Spiro [31]; K o m f e l d and Kornfeld [29]); but in the case of L-PHAP, this heterogeneity appears to be restricted to linkage differences among about 5--10% of the mannose residues. We have presented evidence for the existence of mannose residues substituted at C-3 and C-4 and disubstituted at C-2 and C-3, C-2 and C-4, as well as C-2 and C-6. Some of these products could be biosynthetic intermediates. A similar heterogeneity in the branch point has been f o u n d in an oligosaccharide isolated from the tissues of a patient with fucosidosis [20]. Lis and Sharon [6] interpreted their observed heterogeneity in soybean agglutinin to indicate a mixture of two major forms, namely a: M a n 2 ( a , 1 -~ 2) M a n ( a , 1 -* 6 ) \ Man(H, 1 -~ 4 ) G I c N A c G I c N A c - A s n M a n 4 ( a , 1 ~ 2) M a n ( a , 1 - , 3) /

glycopeptide and a glycopeptide with an additional (a, 1-~ 3/6) branch point in the mannose rich side chain. The major differences between their observations and ours are the fact that they find an average of nine mannose residues per soybean agglutinin oligosaccharide unit and did not report the presence of minor peaks indicating further structural microheterogeneity. With the increased sensitivity of computerized mass-spectrometric and mass-chromatographic techniques, one may assume that such findings of microheterogeneity will become more frequent and will have to be explained in both biological and biosynthetic terms. Despite a number of recent studies indicating special biological roles for glycoprotein oligosaccharide units, the role of carbohydrate in the structure and function of plant lectins remains unclear, since several lectins, most notably concanavalin A, are devoid of carbohydrate. It is of interest that soybean agglutinin (which is not a mitogen) and L-PHAP shown extensive amino acid sequence homology [32] but differ significantly in the size (but not the basic structure) of the oligosaccharide units. More detailed structural analysis of PHAP and other glycoprotein lectins, and studies of the results of enzymatic removal of the terminal residues of the oligosaccharide units on biological activity, may shed more light on this problem.

395 Acknowledgments We wish to thank Dr. Charles C. Sweeley for the use of the mass-spectrometric facility and for his interest during the course of this work. We also wish to acknowledge the advice and assistance of Mr. Jack Harter and Mr. Sun-Sang Sung in obtaining the mass spectra. We are grateful to Dr. Robert Heinrikson for his interest in and encouragement of this project. This work was supported in part by American Cancer Society Institutional Grant No. In-41-0; the Louis Block Fund for Basic Research and Advanced Study Grant No. 26, U.S.P.H.S. Grants HD°06426, HD-04583, and AM-05996, and the National FoundationMarch of Dimes 1-340. G.D. is the recipient of Research Career Development Award KO4 NS00029. The Franklin McLean Memorial Research Institute is operated by The University of Chicago for the U.S. Department of Energy under Contract No. EY-76-C-02-0069. References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 26 29 30 31 32

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