Structure of sialylated fucosyl lactosaminoglycan isolated from human granulocytes.

Sialylated fucosyl lactosaminoglycan was isolated from human neutrophilic granulocytes and its structure was elucidated. The lactosaminoglycan glycopeptides were digested by endo-beta-galactosidase and "the core portion" and released oligosaccharides were analyzed by permethylation, fast atom bombardment mass spectrometry, and exoglycosidases. In addition, lactosaminoglycan saccharides were obtained by hydrazinolysis and the structures of fractionated sialyl oligosaccharides were analyzed by fast atom bombardment mass spectrometry and permethylation coupled with exoglycosidase treatment. The structure of one of the major components was found to be: (Formula: see text). This structure is unique in that 1) four linear polylactosaminyl side chains are attached to the core portion, 2) the side chain arising from position 4 of 2,4-linked mannose contains one or more alpha 1----3 fucosyl residues, 3) the side chain arising from position 6 of 2,6-linked mannose is terminated with NeuNAc alpha 2----3Gal(Fuc alpha 1----3)GlcNAc, sialyl Lex, and 4) the side chain arising from position 2 of 2,4-linked mannose is terminated with sialic acid through alpha 2----6 linkage.

Polymorphonuclear neutrophils or neutrophilic granulocytes are playing a significant role in nonspecific antibodyand complement-directed host defense. The cell-surface structures of neutrophils are believed to participate in an array of neutrophil functions, such as adherence to vascular endothelium, chemotaxis, ingestion, and microbial killing. In particular, exposure of neutrophils to chemotatic factors induces ruffling of their plasma membrane, increases their surface area, changes their shape from spherical to polarized, decreases their negative net surface charge, and increases their adherence to endothelial cells (1). Decreased neutrophil adherence and chemotaxis have been associated with congenital absence of a membrane glycoprotein in a patient with recur-Lactosaminoglycan rent infections (2). Furthermore, knowledge of the cell-surface antigens of granulocytes is important for transfusion of leukocytes and in the analysis of hematological disorders in the myeloid (granulocyte-monocyte) cell lineage. Despite the importance of granulocyte cell-surface components, systematic studies on membrane glycoproteins of granulocytes have not been made.

Structures of Sialosyl Saccharides Obtained from Gpa-
Based on the results described in the "Miniprint," the partial structure of sialosyl lactosaminoglycan from human granulocytes can be summarized as follows.
In order to more fully characterize the sialylated glycopeptides which appeared to contain a mixture of sialylated saccharide chains, glycopeptide a and glycopeptide b were subjected to hydrazinolysis (9,19) and saccharides thus obtained were subjected to quaternary aminoethyl (QAE)-Sephadex column chromatography as described (3,20). As shown in Fig.  5, the saccharides from Gpa were separated into five fractions and A-1, A-2, A-2', and A-3 were recovered as sialylated saccharides. Gpb similarly produced four fractions of sialylated saccharides. These fractions, A-1, A-2, A-2', and A-3, were found to be mono-, di-, di-, and trisialylated saccharide based on the determination of sialic acid and hexose content. Since sialosyl saccharides from Gpb were not available in sufficient amounts, only the saccharides from Gpa were analyzed further.
Structure of A-I-Permethylation analysis indicates that A-1 is composed of 12 lactosaminyl units and contains 1 mol of a 2 4 -l i n k e d sialic acid and 3 mol of nonreducing terminal galactose (Table IV). The same analysis indicates that A-1 contains three fucosyl residues attached to polylactosaminyl side chains based on the amount of 6-0-methyl-N-acetylglucosamine. As shown previously, fucose is bound to C-3 of Nacetylglucosamine to form the Gal/3l4(Fuca1+3)GlcNA~ structure (3). Fast atom bombardment mass spectrometry of permethylated A-1 (Fig. 6A) provided a series of signals which are derived from lactosaminyl chains and these are summarized in Table V. These signals apparently are derived from 'Portions of this paper ("Materials and Methods," portions of "Results," Fig. 1  Saccharides obtained by Gpa and Gpb were applied to a column (0.9 X 8 cm) of QAE-Sephadex equilibrated with 2 mM Tris-HCI, pH 8.0. After washing with 15 ml of the same buffer, the elution was carried out with a linear gradient of 5 mM Tris-HC1, pH 8.0, to 100 mM sodium phosphate buffer, pH 7.0 (50 ml of each solution). The column was further washed with 200 mM sodium phosphate buffer, pH 7.0. Fractions of 0.75 ml were collected and aliquots were taken for determination of sugars by the anthrone reaction. A , saccharides from Gpa; B, saccharides from Gpb.    indicates that fucose is bound to HexNAc residue rather than Hex, since HexNAc residues bearing methoxyl at the C-3 position lose methanol very strongly (9,10). Combining the results obtained by FAB-MS2 with permethylation studies, it was concluded that the structure of A-1 has four different side chains (see below also): one without any substitution, one terminated with 2-6-linked sialic acid, and the other two with various degrees of fucosylation (see Fig. 7). In order to confirm the above conclusion and elucidate the distributions of these side chains, A-1 was digested extensively with a mixture of jack bean 0-galactosidase and P-Nacetylglucosaminidase or with a mixture of clostridial neuraminidase, jack bean @-galactosidase, and P-N-acetylglucosaminidase. The products were then permethylated and the following results were obtained. The first sample produced  Table V.   Fig. 7. Structure of A-2-Permethylation analysis indicates that A-2 is composed of 12 lactosaminyl units and contains 1 mol each of a 2 4 -l i n k e d sialic acid and a2+3-linked sialic acid and four fucosyl residues attached to polylactosaminyl side chains (Table IV). FAB-MS of permethylated A-2 ( Fig. 6 B ) afforded a series of signals of the same m/z values as those obtained from A-1. Additional signals were present at: m/z 999,1448, and 1897 which correspond to the fragment ions of composition of NeuNAc . Fuc . Hex. HexNAc+, NeuNAc . Fuc . Hexa. HexNAc2+, and NeuNAc . Fuc . Hexs. HexNAc3+ ( Table   V). As shown previously (3), sialic acid is bound to galactose and fucose is bound to N-acetylglucosamine. These signals, therefore, suggest the presence of Structure 3, Structure 4, and Structure 5 (Table VI) in the side chain of A-2. Since these fragments can be observed only from sialyl saccharides which were shown by methylation analysis to contain the a2-3 linkage, such as A-2 and A-3 (see Tables IV and V and Fig. 6), it is likely that the sialic acid in this structure is a2-3-linked. This conclusion is supported by the analysis of oligosaccharides released with endo-@-galactosidase treatment as described in the "Miniprint" (see Table 111). The same analysis also indicated that Structure 3 is a major component and Structure 4 is a minor one, whereas Structure 5 is absent. These combined results therefore indicate the presence of the following structures: N e u N A c~2~3 G a l~1~G 1 c~A c~l~3 G a l~l~G l c N A c~l~3 G a l~l 4 G l c N A  It is noteworthy that this sialylated fucosyl side chain contains only one fucose since no sialylated chain, which contains two or more fucose residues, was detected by FAB-MS (Table V).

NeuNAc-O-Gal-O-GlcNAc~O-Gal-O-GlcNAc~O-Gal-O-GlcNAc~O-
On the other hand, the side chain, which contains two or more fucose residues but no sialic acid, was revealed by FAB-MS (Table V). Particularly, the presence of m / z 1884 indicates the presence of the following structure (Structure 6 in Table  VI) as a component of one of the side chains: Fu tff tff tff A portion of this side chain lacks one or two fucosyl residues as judged from the signals at m/z 1710 and 1536. This conclusion is also based on the fact that only one fucosylated side chain without sialic acid is present in A-2 as shown below. Thus, this side chain has various degrees of fucosylation and its structure can be one of Structures 6-12 in Table VI. Since only a limited amount of A-2 was available, it was not determined which structures are actually present among Structures In order to elucidate the distribution of sialic acid and fucose among the four side chains, A-2 was digested with various combinations of exoglycosidases in the same way as A-1. These experiments indicate that the side chain without any substitution arises from position 2 of 2,6-linked a-mannose, and the side chain sialylated through 2 4 linkage and which has no fucose arises from position 2 of 2,4-linked amannose. In addition, A-2 was digested extensively with jack bean @-galactosidase, (3-N-acetylglucosaminidase, and almond emulsin a-fucosidase (1-+3/4-specific) and this derivative was permethylated. This sample produced 2,3,4-tri-O-methylmannose and 3,4,6-tri-O-methylmannose, indicating that 2-3linked sialic acid is present in the side chain arising from position 6 of 2,6-linked a-mannose (see Fig. 7).
Structure of A-2'"Permethylation analysis of A-2' indicates that it consists of 9 lactosaminyl units, two sialic acid residues (2-3-and 2&-linked), and three fucosyl residues attached to N-acetylglucosamine in the polylactosaminyl side chains. FAB-MS afforded a similar series of signals to that observed in A-2 (data not shown). The results indicate that the structure of A-2' is similar to A-2 except that the fucosylated side chain is shorter and contains less fucose. Further analysis of A-2' was not made in the present study due to the limited amount available. Structure of A-3-Permethylation analysis of A-3 indicates that A-3 consists of 8 lactosaminyl units, three sialic acid residues (one 24-linked and two of 2+3-linked), and two fucosyl residues in polylactosaminyl side chains. FAB-MS of permethylated A-3 provided a similar series of fragment ions to that obtained from A-2 (Table V, Fig. 6C). A-3, however, 7-12.
did not produce m/z 1362 for Hex3. HexNAc3+ or its fucosylated derivatives nor was m/z 1261 for Fucz. Hexz.HexNAcz detected. These results indicate that the structure of the nonsialylated chain is:

Gal~l-riGlcNAc~l~3Gal@14GIcNAc~l~Man
To elucidate the distribution of sialic acid, A-3 was digested with P-galactosidase, @-N-acetylglucosaminidase, and a-fucosidase. The product was permethylated and the permethylated sample produced 3,4,6-tri-O-methylmannose with the concomitant loss of 3,6-di-O-methylmannose. Assuming that 2 4 -l i n k e d sialic acid is present in the same side chain as A-2, the structure of A-3 can be proposed as shown in Fig. 7. A part of the side chains arising from C-2 of 2,6-linked mannose are probably fucosylated since the signal (m/z 999) for NeuNAc .Fuc. Gal. GlcNAc is stronger in A-3 than that in the A-2 sample. In order to cocfirm this conclusion, A-3 was digested with neuraminidase, @-galactosidase, and 8-N-acetylglucosaminidase. The permethylated sample showed that only a part (0.6 mol) of the chain arising from position 2 of 2,g-linked mannose was removed by this treatment, indicating that 40% of the side chain contains fucose. Since A-3 consists of 8 lactosaminyl units as a total, the total number of lactosaminyl units in three sialylated chains should be 6. However, a portion of each sialylated side chain contains 3 lactosaminyl units as judged from FAB-MS. These combined results indicate that the number of lactosaminyl units in sialylated side chains varies from 1 to 3 (i.e., m, n, o in Fig. 7 = 0 to 2).
Fucosyl Lactosaminoglycan Glycoproteins in Granulocytes- Fig. 8 shows the profile of the granulocyte membrane proteins detected by immunostaining using monoclonal anti-stagespecific embryonic antigen (SSEA-I) antibody. Glycoproteins, which carry lactosaminoglycan with Gal@l+4(Fucal-, 3)GlcNAc structure, should be reactive with this antibody (11) and three glycoproteins with molecular weights of 130,000-170,000, 100,000, and 70,000 were revealed. Therefore, these glycoproteins are concluded to be the carriers of fucosyl lactosaminoglycan. Since sialylated fucosyl lactosaminoglycan contains the Gal@1+4(Fuc~1+3)GlcNAc terminal structure, it is likely that these glycoproteins also carry the sialylated fucosyl lactosaminoglycan. These glycoproteins were, however, poorly recovered in the extracts of 1% Triton X-100 or 0.5% Nonidet P-40, which is generally used for extraction of plasma membrane proteins (data not shown). Solubilization of these lactosaminoglycan-glycoproteins was achieved by using 0.2% SDS, which in general enables us to extract cytoskeletal proteins and cytoskeleton-associated proteins (22).

6)Man~l+4GlcNAc~l+4(+Fucal-&)GlcNAc+Asn.
Interesting results were obtained by FAB-MS on the length of each side chain. A-2 showed the fragment ions m/z 1710 for Fuc2 Hex3. HexNAcs and 1884 for Fuc~. Hexs. HexNAc3, whereas A-1 lacked the ion of 1884 and A-3 lacked both 1710 and 1884 (Table V, Fig. 6). These differences in fucosylated side chains revealed by FAB-MS correlate well with the results obtained by permethylation analysis. A-2 contains the highest number of fucose residues linked to lactosaminyl units, whereas A-3 contains the smallest number of fucose and total lactosaminyl units (Table IV). These results lead us to conclude that the fragment ions afforded by FAB-MS are reliably indicative of the relative lengths of different side chains present in sialyl saccharides. However, it is not possible at this point to conclude whether the quantitative differences in the intensities of different signals obtained by FAB-MS actually reflect the amount of the structures indicated. This is because we do not know whether the abundance of these ions is affected by their composition. On the other hand, different intensities of the same signal among different samples probably reflect the actual amount of the indicated structure, since the same chemical structures are compared. The use of FAB-MS is indispensable for the analysis of lactosaminoglycan, since it provides sequence information on all side chains of the glycopeptides, which includes the nonreducing terminal portions, and defines the minimum length of long carbohydrate chains.
It is significant that the NeuNAccu24Gal terminal is present in the polylactosaminyl side chain arising from position 2 of 2,4-linked a-mannose, whereas the NeuNAca2+ 3Gal terminal is mainly present in the polylactosaminyl side chain arising from position 6 of 2,g-linked a-mannose. A similar selective location of sialic acid has also been observed in other glycoproteins (9,23) although, to our knowledge, the present work is the first report on locations of sialic acid in tetraantennary glycopeptides. Furthermore, fucosylation at N-acetylglucosamine preferentially takes place in polylactosaminyl side chains arising from position 4 of 2,4-linked mannose and position 6 of 2,6-linked mannose (Fig. 7). A similar selective distribution of fucosyl residues in the side chains was reported in the glycopeptide structures of human al-acid glycoprotein (24) and carcinoembryonic antigen (25), although these glycoproteins mostly contain only l lactosaminyl unit in each side chain. It is rather surprising that such preferential sialylation and fucosylation takes place even in long lactosaminyl side chains of granulocyte lactosaminoglycan. and M, -100,000 carry fucosylated lactosaminoglycan. These glycoproteins might be either rich in the hydrophobic peptide portion or associated with cytoskeletal proteins since the efficient extraction of these glycoproteins was achieved only when 0.2% SDS was included. It is interesting to note that Band 3 glycoprotein, which carries lactosaminoglycan in erythrocytes (9, 10, 26), also associates with cytoskeletal protein, spectrin through ankyrin (27). It has been shown 1 2 3

-68K
FIG. 8. Immunoblotting of fucosyl lactosaminoglycan glycoproteins present in human neutrophilic granulocytes. Neutrophil protein extract (160-40 pg of total protein) was separated by 8% polyacrylamide gel in the presence of SDS as described (12). Proteins were transferred to a nitrocellulose sheet and glycoproteins containing the G a l S l~( F u c a l~3 ) G l c N A c antigenic structure were detected by reaction with anti-SSEA-1 monoclonal antibody followed by rabbit anti-mouse immunoglobulin serum and '?-protein A. Lane 1, 160 pg of total protein; Lane 2, 80 pg of total protein; Lane 3,40 pg of total protein from SDS extract. The numbers at the side indicate the molecular weights estimated by standard proteins. The same result was obtained by anti-PMN-6 antibody. that a glycoprotein carrying lactosaminoglycan gives a characteristic broad band on polyacrylamide gel electrophoresis (9,26,28,29), probably due to the heterogeneity of highmolecular weight carbohydrates. Immune reactive glycoprotein bands shown in the present study also gave broad bands, which is consistent with the property of the glycoproteins carrying lactosaminoglycan. It has been reported that decreased neutrophil adherence and chemotaxis in a patient have been associated with congenital absence of the membrane glycoprotein with M, -150,000 (30). It is of interest to know if this glycoprotein is the same as the fucosyl lactosaminoglycan glycoprotein detected by our studies. The present studies show that the sialyl lactosaminoglycan of human granulocytes contains a significant amount of NeuNAccu2+3Gal~1+4(Fuc~1+3)GlcNAc, sialyl Le' structure? This structure was previously reported in rat brain glycoproteins (31), but not in glycoproteins from human tissue. The isomer of the sialyl Le' structure is NeuNAca2-3Ga1~1+3(Fuca1+4)GlcNAc, sialyl Le" structure, and the presence of sialyl Le" has been reported in human gastrointestinal tumor cells and fetal cells (32). The same structure, however, is absent in normal gastrointestinal cells. Prieels et at. have shown (33) that a fucosyltransferase purified from human milk transfers fucose to both Galpl+3GlcNAc (type I) and Galpl4GlcNAc (type 11) structures, which results in the formation of the Galp1+3(Fuccul+4)GlcNAc (Le") or Gal~l+4(Fucal-3)GlcNAc (Le") structure. On the other hand, Sheares and Carlson showed that GalpldGlcNAc and Gal@l+3GlcNAc are formed by different galactosyltransferases (34). Therefore, the formation of either Le" or Le' de- The definition of Le' is according to Urdal et al. (8). thus it is the same as the X-hapten or lacto-N-fucopentaose I11 structure, Gal61-4(Fucal+3)GlcNAc. The blood group which is present on the Le"-Le"-individual was once called Le', but is not related to the Xhapten described in this study.
Lactosaminoglycan pends on which of the different backbones, type I or type 11, is formed by a specific galactosyltransferase. Similarly, an cu2-3 sialyltransferase extensively purified from rat liver transfers sialic acid to both Gal@14GlcNAc and Gal@1+ 3GlcNAc (35). However, the a1+3/4 fucosyltransferase mentioned above cannot transfer fucose to NeuNAccu2+3Gal@l+ 3/4GlcNAc (33), nor can the a 2 4 3 sialyl transferase sialic acid to Gal@l~3/4(Fuca14/3)GlcNAc (35). Human granulocytes, gastrointestinal tumors, and fetal tissues must, therefore, have a fucosyltransferase or a sialyltransferase with a broader substrate specificity than the transferases so far purified. It will be interesting to see which of these transferases has the required specificity in these tissues.
Our present and previous studies (3) have yielded detailed structures of granulocyte lactosaminoglycan. We have previously postulated that the amount of lactosaminoglycan parallels the degree of maturation of human myelocytic leukemia cells; immature blastoid cells contain a negligible amount of lactosaminoglycan, whereas promyelocytic leukemic cells express a significant amount of lactosaminoglycan (29). In contrast, Van Beek et al. reported that cancer-related glycopeptides with high-molecular weights are present in leukemic cells and these glycopeptides were not diminished after in vitro differentiation of these cells (36). Since our previous work is based on cell surface labeling followed by endo-@galactosidase treatment and the studies of Van Beek et al. are based on metabolic labeling, neither method provides structural information on a sound chemical basis. It will be important to chemically analyze glycopeptides obtained from leukemic cells at various stages of maturation in order to understand lactosaminoglycan expression in differentiation and leukemogenesis. The majority of ratelid15 and methods a r e the ram as described i n the previous papers (3,9.101 except the fallaing: