Identification and structural characterization of Lyt-1 glycoproteins from tunicate hemocytes and mouse thymocytes

Comp. Biochem. PhysioLVol. 99B, No. 4, pp. 741-749, 1991 Printed in Great Britain

0305-0491191 $3.00+ 0.00 © 1991 PergamonPressplc

IDENTIFICATION A N D STRUCTURAL CHARACTERIZATION OF LYT-1 GLYCOPROTEINS FROM TUNICATE HEMOCYTES A N D MOUSE THYMOCYTES H O D A I.NEGM,* M O H A M E D H. MANSOUR and EDWIN L. COOPER~'~ i'Department of Anatomy, School of Medicine, Universityof California,Los Angeles,C A 90024, U S A (Received I0 December 1990)

Abstract--1. A panel of monoclonal antibodies specific to murine Lyt-1 allotypic and framework determinants was used to investigate the possible occurrence of a Lyt-1 homolog in tunicate (protochoradte) hemocytes. 2. In immunopreeipitation experiments, antigenic activities were associated with a major 67 kDa component on tunicate hemocytes and C57B1/6 mouse thymocytes. 3. Tunicate and mouse Lyt-1 molecules were compared, in terms of glycosylation, by their sensitivity to giycosidases and analyses on one- and two-dimensional gel eleetrophoresis. 4. Each of the two molecules appeared to bear two N-linked oligosaccharides, one high-mannose and one complex-type glycan. 5. Both molecules revealed charge microheterogeneity with differences in sialic acid content accounting for the charge difference between each other. 6. However, the difference in the glycans did not account for the microheterogeneity within each molecule, suggesting that other post-translational modifications might be responsible. 7. At the polypeptide level, comparisons of chymotryptic and endoproteinase-Arg-C peptide maps, as well as CNBr-cleavage products, suggested that tunicate and mouse Lyt-1 molecules are structurally similar and that each may contain at least one intra-chain disulfide bridge. 8. The significance of these findings is discussed in terms of the possible biological role of Lyt-I giycoproteins at different levels of evolution.

INTRODUCTION

T cell differentiation antigens, defined by alloantisera or monoclonal antibodies, have been used extensively to delineate functionally-distinct subpopulations of routine thymic and peripheral lymphocytes (Cantor and Boyse, 1977; Ledbetter and Herzenberg, 1979; Ledbetter et aL, 1980). Recent studies have shown that many of these T cell surface structures are intimately involved in T cell functions, including antigen recognition, and are not solely markers of unique functional subsets (Swain, 1983; Walker et aL, 1984). Among these cell surface components, Lyt-1 has been classically considered a marker for the helper/inducer T cell subset (Cantor and Boyse, 1975). Later studies, however, have revealed a broader tissue distribution for this antigen which is detectable on virtually all T cells (Ledbetter eta[., 1983), a subset of normal B cells (Manohar et aL, 1982) as well as certain tumor B cell lines (Wang et al., 1980; Lanier, 1981). *H.I.N. is the recipient of a doctoral fellowship from the Missions Department, Ministry of Education, Egypt. JiAuthor to whom all correspondence should be addressed. Abbreviations used: Endo-Arg: endoproteinase Arg-C; Endo-F: endo-fl-N-acetylgiucosaminidase F; Endo-H: endo-fl-N-acetylgiucosaminidase H; IEF: isoelectric focusing; NP-40: Nonidet P-40; PMSF: phenylmethylsulfonyl fluoride; SDS-PAGE: sodium dodecyl sulfatepolyacrylamidegel electrophoresis; TCA: trichloroacetic acid.

Relatively limited information regarding the structural and functional characteristics of Lyt-1 is available. The molecule has been characterized as a 67 kDa cell-surface glycoprotein, with a 6 0 k D a protein moiety expressed as a single polypeptide without intermolecular disulfide bridges (Durda et al., 1978; Ledbetter et al., 1981; Tung et al., 1984). Although the biological significance of this molecule is not yet clearly understood, recent studies have indicated augmenting properties of anti-Lyt-I antibodies on alloantigen or lectin-induced lymphocyte proliferation (Hollander et al., 1980, 1981), suggesting a possible role for Lyt-1 in optimizing interleukin 1-mediated lymphocyte activations (Lrgdberg and Shevach, 1985). An evolutionary approach study to the Lyt-I molecule can contribute much to understanding its nature and significance. This has been achieved with respect to Thy-1 which is a classical T cell marker in mice (Mansour and Cooper, 1984). In this study, we report on the identification and structural characterization of Lyt-1 molecules from tunicate hemocytes and mouse thymocytes, and on the structural homology between tunicate and murine Lyt-I glycoproteins. Our findings suggest that many characteristics of the Lyt-1 structural gene had already evolved at the time of the emergence of tunicates. This may contribute to understanding the possible biological significance of Lyt-I glycoprotein at different levels of evolution.

741

HODA I. NEGMet al.

742 MATERIALS AND METHODS

Animals Adult Styela clara (protochordate species of solitary tunicates) were collected and maintained as described previously (Mansour and Cooper, 1984). Male and female, 4-6 weeks old, C57BL/6 mice were purchased from the Jackson Laboratories (Bar Harbor, Maine). Antibodies and chemicals Monoclonal antibody (mAb) anti-mouse Lyt-l.1 (clone 41-16.5, class IgM) and anti-mouse Lyt-l.2 (clone V2-2.1, class IgG 2a) were generously provided by Dr. F. W. Shen, Memorial Sloan-Kettering Cancer Center, NY. (mAb) Rat and anti-mouse Lyt-1 [anti-framework determinant, clone 53-7.3, IgG2a (mAb 53-7.3)] was purchased from Becton Dickinson (Mountain View, California). Purified IgG fractions of goat anti-mouse IgM and mouse anti-rat IgG 2a were from Hybritech Inc. (San Diego, California). All chemicals, except where noted, were reagent grade from Sigma Chemical Co., St. Louis, Missouri. Carrier-free Na~251 with spec. act. of 100 mCi/ml was purchased from Amersham Corp., Los Angeles, California. Immobilized lactoperoxidase-glucose oxidase reagents (Enzymobeads) and Bio-Gel P-6 DG were from Bio-Rad Laboratories, Richmond, California. Purified Endo-/Y-N-acetylglucosaminidase F (from Flavobacterium meningosepticum, 600/z/mg, Endo-F), Endo-/LN-acetylglucosaminidase H (from Streptomyces lividans, 25#/mg, Endo-H), neuraminidase (from Clostridium perfringens, 1 #/mg, Nase), ~-chymotrypsin (from bovine pancrease, TLCK-treated, 50/1/mg) and endoproteinase Arg-C (from mouse submaxillary gland, 250/z/mg, Endo-Arg) were purchased from Boehringer Mannheim Biochemicals (Indianapolis, Indiana). CNBr-Activated Sepharose 4B and mol. wt standards were from Pharmacia Fine Chemicals (Uppsala, Sweden). Ampholines and gel electrophoresis reagents were from LKB (Uppsala, Sweden). Cell-surface radiolabeling and immunoprecipitation Tunicate hemocytes and C57BL/6 mouse thymocytes were collected separately, washed and adjusted to 1 × l0 s cells/ml of azide-free tunicate saline (20 mM HEPES/NaOH, pH 7.2 49mM MgSO4 7H20, 11 mM CaCI2, 490 mM NaCI) and 0.2 M phosphate buffered saline, pH 7.2, respectively. Aliquots of I × 10s ceils were surfacelabeled with l mCi NanSI by the Enzymobead-catalyzed reaction as described (Mansour and Cooper, 1984). Cells were washed twice and extracted in lysis buffer [2% nonidetP-40 (NP-40), 20 mM Tris-HCl, pH 8.0, 150 mM NaCI, l mM MgCI2 and 0.I mM phenylmethylsulfonyl fluoride (PMSF)] by incubation on ice for 1 hr. Lysates were centrifuged at 3000 g for 15 min to remove Enzymobeads and subcellular debris. Labeled cell-surface proteins were freed of unbound iodide by gel filtration on a Bio-Gel P-6 DG column equilibrated and eluted with lysis buffer containing 0.5% deoxycholate. Lysates (1001) were pre-cleared with 751 10% IgGsorb for at least 1 hr at 4°C and immunoprecipitated with 5-10 1 of mAbs (unabsorbed or preabsorbed with 5 × 107 mouse thymocytes) for 16 hr at 4°C followed by 501 IgGsorb for an additional 1 hr at 4°C (Negro and Cooper, 1985). For mAb 41-16.5 and mAb 53-7.3 that do not bind to IgGsorb directly, aliquots of IgGsorb were pre-coated with purified IgG fraction of goat anti-mouse IgM and mouse anti-rat IgG 2a, respectively, and washed twice before addition to cell lysates. Immunoprecipitates were washed three times (Kessler, 1975) in wash buffer (1 mg/ml ovalbumin, 650 mM NaC1, 0.5% NP-40, 50mM Tris-HCl, pH8.0, 5mM EDTA) prior to electrophoretic analysis. Gel electrophoresis and analysis Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was carried out on discontinuous

vertical slab gels (12.5%, 15% or 17.5% acrylamide) using a modified sample buffer (2% SDS, 200 mM Tris-HCl, pH 8.0, 0.1 mM EDTA, 5% glycerol and 5% 2-mercaptoethanol) of Laemmli (Laemmli, 1970). Molecular weight markers were phosphorylase b (94 kDa), albumin (67 kDa), ovalbumin (43 kDa), carbonic anhydrase (30 kDa), trypsin inhibitor (20 kDa) and lactalbumin (14.4 kDa). Samples to be analyzed by two-dimensional gel electrophoresis were separated by SDS-PAGE in the first dimension and by isoelectric focusing (IEF) in the second dimension, according to Shackelford and Strominger (1980). Generally, the tool. wt range 60,000-70,000 was excised horizontally (across the lanes of the SDS gel) using dansylated tool. wt markers to locate the area of interest. The excised gel strips were equilibrated for 1 hr at room temperature in 2% Nonidet P-40 (NP-40), 6 M urea and 0.2% ampholines of pH 9-11. The gel strips were blotted dry and applied at the basic end of an IEF slab gel (11 cm wide, 1 mm thick, 5% polyacrylamide, 8 M urea, 2% NP-40 and 2% ampholines in proportions of 60% pH 3.5-10, 20% pH 6-8 and 20% pH 4 and 6) on an LKB multiphore 2117 horizontal gel apparatus. The samples were focused at 1000 V at 4°C for 7-8 hr and the pH gradient determined using an LKB fiat-surface electrode. Gels were fixed with 0.7 M trichloroacetic acid (TCA), 0.13 M 5-sulfosalicylic acid, 30% methanol for 1 hr then washed with 10% methanol, 7.5% acetic acid. Both SDS and IEF gels were dried and autoradiograpbed at - 7 0 ° C using Kodak XAR-5 film and intensifying screens (Picker International, Norwalk, California).

Glycosidase digestions For Endo-F and Endo-H digestions, equal amounts of mAb 53-7.3 immunoprecipitates from tunicate bemocytes and C57BI/6 mouse thymocytes were dissolved by boiling in 301 of 1% SDS, 1% 2-mercaptoethanol, 20raM Tris-HC1, pH 8.0 These aliquots were mixed either with 100/~1 of 100raM sodium phosphate, pH6.1, 50raM EDTA, 1% NP-40 and 5 #1 (0.5 #) of Endo-F (Elder and Alexander, 1982), or with 100#1 of 50raM sodium citrate-phosphate, pH5.5, 1% NP-40 and 11 (10m/~) of Endo-H (Tarentino and Maley, 1974). The samples were incubated for 18hr at 37°C, precipitated with 50/zl TCA and 1 ml acetone at -20°C for 1 hr, washed with I ml cold acetone and dried under nitrogen gas before analysis by SDS gel electrophoresis and IEF. For neuraminidase treatments, sample aliquots of 100/~1 were adjusted to pH 5.5 by addition of 1 vol 50 mM sodium acetate, pH5.5, 150 mM NaC1, 1 mM CaCI2, 0.5% NP-40. Neuraminidase (1/~/ml of acetate buffer, pH5.5) was treated with 2raM PMSF and aprotinin (at 0.5-1 trypsin inhibitor unit/ml, Sigma) for 45 rain on ice to inhibit proteolytic activity and added in 100/~1 aliquots to the samples. The digestion proceeded for 4hr at 37°C and the samples acetone-precipitated and analyzed by SDS gel electrophoresis and IEF. Control samples were treated similarly but in the absence of the digestive enzymes. Limited proteolysis and peptide mapping Partial proteolysis of proteins was carried out with ct-chymotrypsin or Endo-Arg and two one-dimensional peptide mapping procedures were utilized for analysis. The first procedure was carried out according to the method of Cleveland et al. (1977). Briefly, equal amounts of mAb 53-7.3 immunoprecipitates were non-treated or digested with Endo-F and separated on a 12.5% polyacrylamide slab gel. Areas of the gel with the relevant mol. wt (located using dansylated mol. wt markers) were excised, equilibrated for 30 min at room temperature in 125 mM Tris-HCl, pH 6.8, I mM EDTA, 0.1% SDS, 5% 2-mercaptoethanol, applied to a 17.5% polyacrylamide gel and overlaid with the same buffer containing ~-chymotrypsin or Endo-Arg (2.5 g/gel slice). The samples were run into the stacking gel, paused for

743

Structural conservation of Lyt-1 glycoproteins 30 min and allowed to proceed until the dye-front reached the bottom of the resolving gel. The gels were processed as described above. In the second procedure, equal amounts of non-treated or Endo-F digested samples were separated on 12.5% polyacrylamide gels and the areas of interest located and excised as above. Gel slices were diced into 1 mm cubes and eluted in lml of 0.1% SDS at 37°C for 12hr. Eluates were collected by passage through nylon wool, and lyophilized to dryness. They were then dissolved in 150/~l of 50raM ammonium bicarbonate, pH 7.7 containing 5 g of ~-chymotrypsin or Endo-Arg and 100 g of BSA, flushed with nitrogen gas and incubated at 37°C for 12 hr. The digestion was halted by freezing and lyophilization to dryness. Lyophilized samples were dissolved in 6 M urea, 2% NP-40, 0.2% ampholines of pH 9-1 l, applied to the basic end of IEF slab gels and focused at 30 W for 1.5 hr at 4°C. The gels were processed as indicated above.

Cleavage with CNBr Equal amounts of mAb 53-7.3 immunoprecipitates were separated by 12.5% polyacrylamide slab gels and the areas of interest located, excised, diced and eluted as described above. After lyophilization, the samples were dissolved in 150#l of 70% formic acid saturated with CNBr. The reaction mixtures were flushed with nitrogen gas and cleavage allowed to proceed for 24hr at 20°C in the dark. The reaction was stopped by the addition of l0 vols of water and lyophilization, which was repeated thrce times. The lyophilized samples were then dissolved in Laemmli sample buffer and analyzed by SDS-PAGE (15% polyacrylamid¢ under non-reducing and reducing conditions).

RESULTS

Immanoprecipitation of membrane antigens recognized by mAb anti-Lyt-I allotypic and framework determinarlls Tunicate hemocyte and C57BL/6 mouse thymocytes were labeled with 12sI by the lactoperoxidase-glucose oxidase catalyzed reaction. After solubilization of cells, lysates were immunoprecipitated with mAbs anti-Lyt-1 allotypic and framework determinants and IgGsorb as described. Results of SDS-polyacrylamide gel electrophoresis (SDS-PAGE) in 15% gels are shown in Fig. 1. Under non-reducing conditions, both mAb V2-2.1 and mAb 41-16.5 precipitated from tunicate hemocytes a labeled component of 67kDa (Fig. IA, lanes 1 and 6). The same component was also precipitable with mAb V2-2.1 (preabsorbed with CBA/J mouse thymocytes) (Fig. 1A, lanes 3 and 8, respectively). In contrast, absorption of mAb V2-2.1 with C57BL/6 mouse thymocytes (Fig. 1A, lane 4) and mAb 41-16.5 with CBA/J mouse thymocytes (Fig. 1A, lane 7) markedly reduced the quantity of the 67 kDa component detected and resulted in a gel pattern indistinguishable from those obtained with IgGsorb alone (Fig. l, lanes 2 and 5). A high mol. wt component was detected in various amounts with specific as well as control precipitations, thus establishing that both Lyt-l.2 and Lyt1.1 antigenic determinants are associated with 67 kDa polypeptides on tunicate hemocyte surfaces.

A}

B} 1

2

3

4

5

6

7

8

T

.<

94

>.

.<

67

>~

.<

43

~t

30

~t

20

>.

14.4

>.

n - red

M

T

M

red

Fig. 1. SDS-PAGE (15%) of 125I-labeledtunicate hemocytes and C57BL/6 mouse thymocytes immunoprecipitated with different mAb anti-Lyt-1 antigenic determinants. (A) Immunoprecipitates of 12SI-labeled tunicate hemocytes under non-reducing conditions with (1) mAb V2-2.1, (2) IgGsorb, (3) mAb V2-2.1 absorbed with CBA/J mouse thymocytes, (4) mAb V2-2.1 absorbed with C57BL/6 mouse thymocytes, (5) IgGsorb pre-coated with IgG fraction of goat anti-mouse IgM, (6) mAb 41-16.5, (7) mAb 41-16.5 absorbed with CBA/J mouse thymocytes, (8) mAb 41-16.5 absorbed with C57BL/6 mouse thymocytes. (B) Immunoprecipitates of l:SI-labelledtunicate hemocytes (T) and C57BL/6 mouse thymocytes (M) with mAb 53-7.3 under non-reducing (n-red) and reducing (red) conditions. Arrows indicate the positions of mol. wt markers in kDa.

744

HODA I. NEGMet al.

Immunoprecipitates with mAb 53-7.3 from tunicate hemocytes and C578BL/6 mouse thymocytes (Fig. 1B) revealed a major 67 kDa component under non-reducing and reducing conditions (together with minor contaminants, which probably adsorbed non-specifically to the mouse anti-rat IgG2a coating the IgGsorb). This suggests that as in mice, all antigenic specificities associated with the putative Lyt-1 homolog in tunicates coincide with a single macromolecule, which is not disulfide-linked to other polypeptides on the cell surface. The carbohydrate moieties o f tunicate and mouse Lyt-1 molecules are of similar size

The oligosaccharides associated with the tunicate hemocytes and mouse thymocyte Lyt-1 molecules were characterized by their susceptibility to the enzymes Endo-F, Endo-H and neuraminidase. Both Endo-F and Endo-H cleave the linkage in the core of N-linked oligosaccharides, leaving one N-acetylglucosamine residue attached to an asparagine residue of the peptide chain, whereas neuraminidase trims sialic acid residues from N-linked as well as O-linked glycans. Endo-F removes both highmannose and complex glycans, changing size and, possibly, charge of the substrate glycoproteins (Elder and Alexander, 1982), whereas Endo-H removes high-mannose glycans only, thus altering size but not charge (Tarentino and Maley, 1982). Equal volumes of mAb 53-7.3 immunoprecipitates of tunicate hemocytes and mouse thymocytes were incubated with or without the endoglycosidases or neuraminidase as described in the Materials and Methods section and the mobility of the 67 kDa major component analyzed by S D S - P A G E (Fig. 2). The 67 kDa component of both tunicate and mouse was susceptible to both Endo-F and Endo-H treatments. Endo-F and Endo-H digestions converted the 67kDa polypeptide in both tunicate and mouse preparations into 60 kDa and 63 kDa components respectively. The shift of about 4 kDa with Endo-H treatment is consistent with the removal of one highmannose glycan unit from both polypeptides. With Endo-F treatment, the additional reduction of mol. wt by 3000 was indicative of the removal of one T

M

T

M

complex-type glycan, and both tunicate and mouse molecules are apparently associated with two N-linked oligosaccharides; one high-mannose and one complextype. The effect of neuraminidase treatment was not apparent, since it did not result in any alterations in the mol. wts of both tunicate and mouse polypeptides. The tunicate and mouse Lyt-1 glycoproteins differ in their sialic acid content

To investigate further the nature of the glycan sidechains, equal volumes of mAb 53-7.3 immunoprecipitates of tunicate hemocytes and mouse thymocytes were either untreated or treated with endoglycosidases or neuraminidase and analyzed by two-dimensional electrophoresis. A strip encompassing the mol. wt range 67,000-60,000 was excised horizontally across the lanes of the first-dimension SDS gel and applied at the basic end of the second-dimension IEF gel, such that the direction of IEF parallels that of SDS-PAGE. As shown in Fig. 3 (lane NT), both untreated tunicate and mouse Lyt-1 glycoproteins exhibited heterogeneous patterns and focused as six prominent spots. Within the pI range 7.7-7.8, both molecules share a major band (it appears as a doublet), whereas a significant difference in their patterns was noted in the pI range 5.5-6.5. In this range, and in spite of the overlap of pI 5.8 and 6.0, it was clear that the tunicate pattern was slightly more basic than that of the mouse. Treatment with Endo-F (Fig. 3, lane Endo-F) collapsed the tunicate pattern to four spots corresponding to the four most basic bands in the original pattern. This implies that, at least in part, the charge heterogeneity of the tunicate Lyt-1 molecule is due to N-linked glycans. This, however does not apply to the mouse Lyt-1 glycoprotein, since Endo-F treatment yielded the same number of spots as the glycosylated material, but with a shift in the pattern to a more basic pI. Endo-F treatment seemed, thus, to affect both molecules similarly in terms of removal of acidic entities from their complex oligosaccharides. The nature of these acidic residues was tested by inspecting the pattern of the tunicate and mouse Lyt-I molecules after neuraminidase treatment. As shown in Fig. 3 (lane Nase), treatment with T

M

T

M

94>67>

43~-

NT Endo- F Endo-H Nase Fig. 2. Comparison of glycosidase-treated tunicate and mouse Lyt-1 glycoproteins by one-dimensional electrophoresis. Equal volumes of radioactive post-mAb 53-7.3 column fractions of tunicate (T) and C57BL/6 mice (M) were either not treated (NT) or digested with Endo-F, Endo-H or neuraminidase (Nase) and analyzed on a 12.5% SDS-polyacrylamideslab gel. Arrowheads indicate the mobility of mol. wt markers in kDa. Also shown are the estimated mol. wts of deglycosylated materials.

Structural conservation of Lyt-1 glycoproteins T

M

T

M

T

M

745

T

M

9-

.

pH

7.

.

q

NT

Endo-F

Endo_H

Nase

Fig. 3. Comparison of glycosidase-treated tunicate and mouse Lyt-1 glycoproteins by two-dimensional electrophoresis. Equal volumes of radioactive post-mAb 53-7.3 fractions of tunicate (T) and C57B1/6 mice (M) were treated as indicated in the legend of Fig. 3. The strip encompassing the mol. wt range 67,000-60,000 was excised horizontally across the lanes of the first-dimension SDS gel and applied at the basic end of the second-dimension IEF gel, such that the direction of IEF was parallel to that of SDS-PAGE. The samples corresponding to the lanes in Fig. 2 are: not treated (NT), digested with Endo-F, Endo-H or neuraminidase (Nase). The pH gradient was determined by a fiat-surface electrode at 0.5 cm intervals. neuraminidase did not change the number of bands, nor the pI of the tunicate pattern as compared to the untreated material, suggesting that the tunicate Lyt-1 molecule may either lack sialic acid or express substituted sialic acid residues, which are resistant to neuraminidase (Shauer and Faillard, 1968). The latter suggestion may provide a plausible explanation for the disappearance of the two most acidic spots in the tunicate pattern after Endo-F treatment. By contrast, neu~aminidase treatment shifted the mouse pattern towards a more basic pI, without changing the number of spots, as compared to the glycosylated molecule. The resulting pattern was similar to that of the untreated or neuraminidase-treated tunicate Lyt-1 molecule. This observation is consistent with the removal of sialic acid residues from the mouse Lyt-1 molecule and supports the idea that quantitative and/or qualitative differences in sialic acid account for the charge differences between the tunicate and mouse Lyt-1 glycoproteins. As would be expected, after Endo-H treatment, the patterns of both tunicate and mouse molecules were similar to the patterns of their corresponding glycosylated forms in terms of the number of spots and charge (compare lanes NT and Endo-H in Fig. 3), inasmuch as high-mannose oligosaccharides are neutral in charge. It is also noteworthy that in all instances, neither the tunicate nor the mouse patterns were collapsed to a single band. This implies that the microheterogeneity within each Lyt-1 molecule (particularly in the mouse) is unlikely to depend on variations in the N-linked glycans. Either this microheterogeneity represents variations in O-linked glycans, or else chemical modifications such as oxidation, carbamylation or deamidation are responsible.

Structural homology of the tunicate and mouse Lyt-1 polypeptide chains as revealed by peptide mapping To further probe the degree of similarity and/or difference between tunicate and mouse Lyt-1 molecules, equal amounts of mAb 53-7.3 immunoprecipitates of tunicate hemocytes and mouse thymocytes were untreated or digested with Endo-F, separated on a 12.5% polyacrylamide SDS gel and the mol. wt ranges of 67,000 or 60,000 excised and subjected to limited proteolysis with ~-chymotrypsin and Endo-Arg, as described in the Materials and Methods. Comparisons were made utilizing two one-dimensional peptide mapping techniques. By the method of Cleveland et al. (1977), using either ct-chymotrypsin (Fig. 4A) or Endo-Arg (Fig. 4B), the observed patterns of the tunicate and mouse Lyt-1 molecules were similar. All of the 1251 tyrosine-containing chymotryptic peptides derived from both polypeptides were of identical mobility on 17.5% SDS-PAGE. There was however, a difference in the relative intensity in two of the corresponding peptides (broad, fuzzy bands indicated by arrows in Fig. 4A). Although this observation implied that chymotrypsin sites are homologous in the two Lyt-1 polypeptides, it was evident that some of the constituent peptides may structurally vary. The similar peptide patterns obtained after deglycosylation suggested that this variation was not due to size heterogeneity among constituent glycopeptides and indicated that glycosylation sites may be shared between tunicate and mouse Lyt-1 polypeptides. These observations were supported by the Endo-Arg peptide-maps of the two polypeptide chains (aside from some variations in the relative intensity) before and after deglycosylation (Fig. 4B).

746

HODAI. NEGMet al.

A]

B] T

M

NT

T

M

T

Endo- F

M

NT

cl

T

M

Endo. F

O] T

M

T

M

T

M

T

M

pH 7.

6.

5°

NT

Endo- F

NT

Endo- F

Fig. 4. Analysis of the tunicate and mouse Lyt-1 glycoproteins by one-dimensional peptide mapping. Equal amounts of radioactive post-mAb 53-7.3 fractions of tunicate (T) and C57BL/6 mice (M) were not treated (NT) or digested with Endo-F and separated on a 12.5% SDS-polyacrylamide gel. Bands containing 67 kDa (intact) and 60 kDa (deglycosylated)proteins were located and excised as described in the Materials and Methods section. One-dimensional peptide-mapping was either by the method of Cleveland et al. (1977) (25) on a 17.5% SDS-polyacrylamideslab gel (A and B) using intact gel slices or by analyzing digests of proteins eluted from the respectivegel slices by IEF (C and D). In both procedures, materials were digested by ~-chymotrypsin (A and C) or Endo-Arg (B and D) as described in detail in the text. Arrows in A and B point to bands with different intensities in tunicate and mouse patterns. To confirm the structural relationship between the tunicate and mouse Lyt-1 polypeptides, chymotryptic and Endo-Arg digests were compared by one-dimensional IEF (Fig. 4C and D). By this parameter, and at the level of 125I tyrosine-containing chymotryptic (Fig. 4C) and arginine (Fig. 4D) peptides, the structural similarity of the two Lyt-1 polypeptides was apparent. Nevertheless, the differences observed, particularly among the deglycosylated chymotryptic peptide patterns, confirm that in spite of the similarities, the two Lyt-1 polypeptides may not be identical. This observation is complementary to the data obtained with the intact molecules and suggests

that primary structural differences and/or differential post-translational modifications may account for some variations in the structure of the tunicate and mouse Lyt-1 glycoproteins. Comparison o f tunicate and mouse Lyt- 1 CNBr fragments Equal amounts of mAb 53-7.3 immunoprecipitates of tunicate hemocytes and mouse thymocytes were separated on a 12.5% SDS gel and the 67 kDa bands excised and eluted as described in the Materials and Methods section. The eluted fractions were then treated with CNBr and the resulting fragments

Structural conservation of Lyt-I glycoproteins analyzed on a 15% SDS gel under non-reducing and reducing conditions. Under non-reducing conditions, both the tunicate and mouse Lyt-1 molecules were completely cleaved into 30 kDa, 23 kDa and 16 kDa fragments. Analyses under reducing conditions resulted in the disappearance of the 30 kDa fragment from both the tunicate and mouse patterns implying that in this fragment the cleavage products are linked by at least one disulfide bond in both molecules. No additional bands other than the 23 kDa and 16 kDa components were observed, suggesting that the disulfide-linked fragments may consist either of two 16 kDa peptides or a 23 kDa peptide and a peptide of about 7 kDa, which probably eluted in the buffer front, and hence escaped detection. Taken together, the CNBr patterns observed under non-reducing and reducing conditions are consistent with the presence of at least three methionine residues located in similar positions along the tunicate and mouse Lyt-I polypeptides. Furthermore, the data allow the prediction that an intra-chain disulfide bridge is located along the polypeptide, possibly in the same position, in both the tunicate and mouse Lyt-1 molecules. DISCUSSION

The Lyt-1 antigen has, since its discovery in 1968 (Boyse et al., 1968), been useful primarily as a qualitative and quantitative marker in delineating discrete T cell subpopulations (Cantor and Boyse, 1977; Ledbetter and Herzenberg, 1980; Ledbetter et al., 1980). Nevertheless, limited information regarding its structural characteristics, biological significance and distribution in different animal models, is available (Durda et al., 1978; Ledbetter et al., 1981; Tung et al., 1984). In a previous paper, a structural homoiog to the mammalian Thy-1 molecule has been detected on tunicate hemocytes (Mansour and Cooper, 1984), suggesting an early phylogenetic emergence for T cell differentiation antigens. In this present study, several structural criteria were employed to provide evidence for the structural conservation of Lyt-I antigens in tunicates and mice in spite of the wide evolutionary gap. Preliminary evidence for the occurrence of a structural homolog to murine Lyt-1 molecule was investigated using monoclonal antibodies of proven specificity to allotypic and framework determinants associated with this molecule in immunoprecipitation experiments. With either anti-frame work or antiallotypic determinants, the target antigenic determinants are associated on tunicate hemocytes with a single 67 kDa polypeptide chain with no evidence for the presence of multiple forms of the antigen. This observation confirms that, as in mice, all Lyt-1 cross-reacting determinants are co-expressed with the same macromolecule on tunicate hemocytes. The estimated mol. wt of this molecule is in striking agreement with that of the murine Lyt-I molecule (Durda et al., 1978; Tung et al., 1984) as well as its counterpart in humans (Ledbetter et al., 1981) and support the expression of a Lyt homolog on tunicate hemocytes. Further insights on the structural similarities between the 67 kD macromolecule on tunicate hemocytes and the Lyt-I molecule in mice were obtained CBPB ~/4~C

747

using other detailed structural criteria. The association of Lyt-I antigenic determinants with cellsurface glycoproteins on mouse thymocytes was previously documented (Durda et al., 1978; Tung et ai., 1984). In this study, data confirm these observations and suggest further that, in terms of the type and number of glycan units, the tunicate and mouse Lyt-1 molecules are identical. Both molecules express two N-linked oligosaccharides, one of the high mannosetype and one of the complex-type. The demonstration of an Endo-H-sensitive, high-mannose glycan unit on both tunicate and mouse Lyt-1 molecules contradicts the observation of Tung et al. (1984), which suggested the lack of such glycans on the cell-surface expressed mature form of the mouse Lyt- 1. Although the reason for this discrepancy is not clear, and may be due to differences in the mouse strains, our observation is supported by the fact that other mature cell surface glycoproteins including Thy-I (Luescher and Bron, 1985), Lyt-2 (Luescher et al., 1985) and HLA-DRchain (Owen et al., 1981) have also been shown to express high-mannose glycans. After removal of the N-linked glycans, both tunicare and mouse Lyt-1 molecules were resolved into a 60 kD polypeptide equivalent to the 60 kDa biosynthetic precursor previously shown to be devoid of O-linked or N-linked glycans (Tung et al., 1984). This, in turn, suggests that at least in mice, mature Lyt-1 molecules lack O-linked glycans. This may be true for the tunicate Lyt-1 molecule, yet it should be confirmed by examining the biosynthetic pathway. Although the tunicate and mouse Lyt-I molecules contain the same type of N-linked glycans, which seem to account for all the oligosaccharide sidechains, the composition and structure of these sidechains are not identical in the two molecules. This was clearly demonstrated by inspecting two-dimensional gel patterns in which the charge microheterogeneity of both tunicate and mouse Lyt-I molecules was also revealed. Each molecule consisted of six charge variants which, in spite of overlapping at several PIs, were indicative of the slight overall acidity of the mouse Lyt-1 molecule compared to its tunicate counterpart. As revealed by Endo-F and neuraminidase treatments, the difference between the two molecules is attributed to a differential degree of sialylation in both chains and to the presence of additional charged entities in the tunicate Lyt-1 oligosaccharides side-chains. These charged entities were, however, acidic in nature and most probably represent substituted sialic acid residues (e.g. O-acetyiated sialic acid (Schauer and Faillard, 1968) or N-glycolyl neuraminic acid (Carlsson and Stigbrand, 1983)] which escaped neuraminidase digestion, but still contributed to the charge difference between the tunicate and mouse Lyt-I molecules. Although variations in the N-linked glycans account for the charge difference among the tunicate and mouse Lyt-1 molecules, the basis for microheterogeneity within each molecule remains to be fully understood. This heterogeneity has been observed for the murine Lyt-I and human Leu-1/Tl molecules using a different two-dimensional gel system (Ledbetter et al., 1981) and thus, does not appear to be a result of artifactual modifications. Removal of N-linked glycans slightly reduced the heterogeneity in

748

HODA I. NEGM et al.

the tunicate Lyt-1 pattern, still both the tunicate and mouse molecules, each, focused as multiple bands. The potential presence of O-linked glycans and Lyt- 1 aliotypic variants (Mansour and Cooper, 1984) may explain the heterogeneity within the tunicate pattern but certainly not that of the mouse Lyt-1. Alternatively, the most plausible explanation for this phenomenon would be that each polypeptide is subjected to differential post-translational modifications such as oxidation, phosphorylation or deamidation. Interestingly, this explanation implies diversities within the cell population expressing Lyt-1 in tunicate hemocytes and mouse thymocytes and reflects the changes that occur during differentiation, as previously demonstrated by FACS analyses (Ledbetter et al., 1980; Ledbetter et al., 1981; Mansour and CQoper, 1984). The differences in glycosylation between the tunicate and mouse Lyt-I glycoproteins prompted the investigation of the structural relationship between the two molecules at the polypeptide level. Comparison of peptide maps and CNBr-cleavage products strongly suggest that both molecules were closely related in polypeptide structure. In spite of inherent difficulties in gauging the extent of homology by peptide-map comparisons, both molecules appeared to be homogenous in terms of chymotrypsin sites, Endo-Arg-susceptible arginine residues, the location and number of methionine residues and the presence of at least a single intra-chain disulfide bridge. Nevertheless, there appeared to be some charge differences among chymotryptic peptides, indicating that both molecules may be similar but not identical. Definitive statements on the exact structural relationship between the tunicate and mouse Lyt-1 molecules must await the determination of amino acid sequence of both polypeptides. These studies should be particularly informative in determining the size and location of the intra-chain disulfide bridge(s) in both molecules and may ultimately establish the possible structural relatedness of the Lyt-I polypeptide to the immunoglobulin superfamily of molecules, which are involved in different aspects of lymphocyte function (Williams, 1985). The biochemical and serological analyses of Lyt-1 antigens suggest that a highly-conserved polypeptide carries different carbohydrate structures in hemocytes and thymocytes. Although the biological significance of Lyt-1 is still unclear, its molecular properties suggest and/or restrict possible functions in both tunicates and mice. If the polypeptide portion of the molecule mediates its specific function, then this function would probably be the same in both animal models, and the carbohydrate may serve simply to orient the molecule into the cell membrane. The differences in the glycans would then be without major significance and may result from different glycosyl transferases in the different tissues. Alternatively, the polypeptide may serve to: (1) integrate the molecule into the membrane and (2) allow the display of different carbohydrate structures. These glycans could be involved in cell-cell interactions as ligands to be recognized by lectin-like molecules or glycosyl transferases in other cells. Interactions similar to this have been suggested to be involved in controlling tissue organization and self/non-self discrimination in

invertebrates (Parish, 1977) and are in agreement with the proposed role of Lyt-I and its structural homologs in targeting activating signals during lymphocyte differentiation in mammals (Hollander et al., 1980, 1981; Logdberg and Shevach, 1985; Dallman et al., 1984). If Lyt-1 were involved in such functions, the differences in carbohydrate composition between hemocytes and thymocytes may reflect differences in the fine detail of recognition in these two tissues. These ideas should ultimately be tested in functional assays, since they would establish the Lyt-I glycoprotein as a common structure for cell-cell interactions at different levels of evolution. Acknowledgements--We thank Jennifer Wallace and John

Dobak for their excellent help in preparing this manuscript. This work was supported by a grant from the National Science Foundation. REFERENCES

Boyse E. A., Miyazawa M., Aoki T. and Old L. J. (1968)

LyA and LyB: Two systems of lymphocyte isoantigens in the mouse. Proc. R. Soc. Lond. 170, 175-193. Cantor H. and Boyse E. A. (1975) Functional subclasses of T lymphocytes bearing different Ly antigens. 3". exp. Med. 141, 1390-1399. Cantor H. and Boyse E. A. (1977) Lymphocytes as models for the study of mammalian cellular differentiation. lmmunol. Rev. 33, 105-124. Carlsson S. R. and Stigbrand T. I. (1983) Alterations and glycosylation pattern of the Thy-1 glycoprotein during maturation and transformation of mouse T lymphocytes. J. lmmunol. 130, 1837-1842. Cleveland D. W., Fischer S. G., Kirshchner M. W. and Laemmli U.K. (1977) Peptide mapping by limited proteolysis in sodium dodecyl sulfate and analysis by gel electrophoresis. J. biol. Chem. 252, 1102-1106. Dallman M. J., Thomas M. L. and Green J. R. (1984) MRC OX-19: A monoclonal antibody that labels rat T lymphocytes and augments in vitro proliferative responses. Eur. J. Immunol. 14, 260-267. Durda P. J., Shapiro C. and Gottlieb P. D. (1978) Partial molecular characterization of Ly-1 alloantigen on mouse thymocytes. J. Immunol. 120, 53-57. Elder J. H. and Alexander S. (1982) Endo-B-N-acetylglucosaminidase F: endoglycosidase from Flavobacterium meningosepticum that cleaves both high-mannose and complex glycoproteins. Proc. Natn. Acad. Sci. USA. 79, 4540-4544. Hollander N., Pillemer E. and Weissman I. L. (1980) Blocking effect of Lyt-2 antibodies on T cell functions. J. exp. Med. 152, 674-687. Hollander N., Pillemer E. and Weissman I. L. (1981) Effects of Lyt antibodies T-cell functions: augmentation by antiLyt-1 as opposed to inhibition by anti-Lyt-2. Proc. ham. Acad. Sci. USA. 78, 1148. Kessler S. W. (1975) Rapid isolation of antigens from cells with a staphylococcal protein A-antibody absorbent: parameters of the interaction of antibody-antigen complexes with protein A. J. lmmunol. 115, 1617-1624. Laemmli U. K. (1970) Cleavage of structural proteins during assembly of the head of bacteriophage T4. Nature 227, 680-685. Lanier L. L., Warner N. L., Ledbetter J. A. and Herzenberg L. A. (1981) Expression of Lyt antigen on certain murine B cell lymphomas. J. exp. Med. 153, 998-1003.

Ledbctter J. A., Evans R. L., Lipinski M., CunninghamRundles C., Good, R. A. and Herzenberg L. A. (1981) Evolutionary conservation of surface molecules that distinguish T. lymphocyte. J. exp. Med. 153, 310-323.

Structural conservation of Lyt-1 glycoproteins Ledbetter J. A. and Herzenberg L. A. (1979) Xenogenic monoclonal antibodies to mouse lymphoid differentiation antigens. Immunol. Rev. 47, 63-90. Ledbetter J. A., Rouse R. V., Micklem H. S. and Herzenberg L. A. (1980) T cell subsets defined by expression of Lyt-1, 2, 3 and Thy-1 antigens. J. exp. Med. 152, 280-295. L6gdberg L. and Shevach E. M. (1985) Role ofLy I antigen in interleukin l-induced thymocyte activation. Eur. J. Immunol. 15, 1007-1013. Luescher B. and Bron C. (1985) Biosynthesis of mouse Thy-1 antigen. J. Immunol. 134, 1084-1989. Luescher B., Rousseaux-Schmid M., Naim H. Y. MacDonald H. R. and Bron C. (1985) Biosynthesis and maturation of the Lyt-2/3 molecular complex in mouse thymocytes. J. Immunol. 135, 1937-1944. Manohar V., Brown R., Leiserson W. M. and Chased T. M. (1982) Expression of Lyt-1 by a subset of B lymphocytes. J. lmmunol. 129, 532-538. Mansour M. H. and Cooper E. L. (1984) Serological and partial molecular characterization of a Thy-1 homolog in tunicates. Fur. J. Immunol. 14, 1031-1039. Negm H. I. and Cooper E. L. (1985) Evolutionary conservation of Lyt-I alloantigen. Fedn. Proc. 44, 790. Owen M. J., Kissonerghis A. M., Lodish H. F. and Crumpton M. J. (1981) Byosynthesis and maturation of HLA-DR antigens in vivo. J. biol. Chem. 256, 8987-8993. Parish C. R. (1977) Simple model for self-non-self-discrimination in invertebrates. Nature 267, 711-713.

749

Schauer R. and Faillard H. (1968) Das Ver halten N.O.diacetyl-neuroaminsaureglykoside im Submaxillarismucin von Pferd und Rind bei Einwirkung bakterieller Neuraminidase. Hoppe-Seyler's Z. physiol. Chem. 349, 961-968. Shackelford D. A. and Strominger J. L (1980) Demonstration of structural polymorphism among Hla-Dr light chains by two-dimensional gel electrophoresis. J. exp. Med. 151, 144-165. Swain S.L. (1980) T cell subsets and the recognition of MHC class. Immunol. Rev. 74, 129-142. Tarentino A. L. and Maley F. (1974) Purification and properties of an endo a-N-acetylglycosaminidase from Streptomyces griseus. J. biol. Chem. 249, 811-817. Tung J. S., Shen F. W., Deere M. C. and Boyse E. A. (1984) Structure and biosynthesis of Lyt-1 helper/inducer and cytoxic/suppressor subpopulations in mouse and man. Immunogenetics 20, 125-130. Walker I. D., Hogarth P. M., Murray B. J., Lovering K. E., Classon B. J., Chambers G. W. and Mackenzie I.F.C. (1984) Ly antigens associated with T cell recognition and effector function. Immunol. Rev. 82, 47-77. Wang C. Y., Good R. A., Ammirato P., Dymbort G. and Evans R. L. (1980) Identification of a p69, 71 complex expressed on human T cells sharing determinants with B-type chronic lymphatic leukemic cells. J. exp. Med. 151, 1539-1544. Williams A. F. (1985) Immunoglobulin-related domains for cell surface recognition. Nature 314, 579-580.