Characterization of Novel Amino Acid Fucosides*

The structures of FL4b and of two other related amino acid fucosides have been determined by a com-bination of methylation analysis and enzymatic diges- tion. Additionally, the anomeric configurations of the carbohydrate moieties of FL4a, previously shown to be glucosyl(1 -+ 3)fucosyl 1 + threonine (Steiner, S., Via, D. P., Klinger, M., Larriba, G., Sramek, S., and Laine, R. (1978) in Glycoproteins and Glycolipids in Disease Proc- esses (Waiborg, E. F., Jr., ed) pp. 378-403, American Chemical Society, Washington, D. C.) have been deter- mined by enzymatic digestion. The results indicate that the structures are: FL3a, fucosylal -+ threonine; FL3b, fucosylal + serine; FUa, glucosylpl-+ 3fucosylal+ threonine; and FL4b, glucosylpl-+ 3fucosylal+ serine. FL4a, which appears to have the same structure as a component from human urine (Hallgren, P., Lundblad, A., Svensson, S. J. Biol. Chem. 250, 5312- 5314), and FL4b are highly unusual in that they contain fucose in a nonterminal position. The fucosyl-serine linkage found in compounds FL3b and FL4b is a novel structure.


5314), and FL4b are highly unusual in that they contain fucose in a nonterminal position. The fucosyl-serine linkage found in compounds FL3b and FL4b is a novel structure.
Previous studies in this laboratory have demonstrated that radioisotopically labeled fucose is incorporated into a series of low molecular weight compounds (1,2) in normal rat cells.' It was also shown that the level of one of these components, FL4a, is markedly reduced in NRK cells transformed by murine sarcoma virus. Respess and co-workers demonstrated that a lower ratio of FCZ to FL3 could be positively correlated with an increased tumorigenicity of cloned HSV-transformed rat cells.' In addition, changes in the ratio of FL4a to FL3a have been demonstrated to be positively correlated with alterations in the cellular morphological phenotype. For example, butyrate-induced cellular flattening of MSV-NRK cells to a more normal phenotype is accompanied by an increase in the level of FL4a relative to FL3a (1). The connection between butyrate-induced changes in FIAa/FL3a and "oncogenicity" has not been established.
We have previously determined the amino acid and carbohydrate composition of four of these low molecular weight compounds obtained from rat tissues (4). The compositions ' R. A. Respess, L. S. Kucera, and M. Waite, personal communication.

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were found to be: FL3a, fucose:threonine, 1:l; FL3b, fucose: serine, 1:l; FL4a, glucose:fucose:threonine, 1:l:l; and FL4b, glucose:fucose:serine, 1:l:l. By gel filtration chromatography, the apparent molecular weight of FL3a and FL3b was estimated to be approximately 250, and the apparent molecular weight of FL4a and FL4b was estimated to be approximately 500 (4). These results are consistent with the interpretation that FL3a and FL3b have 1 mol each of fucose and amino acid and that FL4a and FL4b have 1 mol each of glucose, fucose, and amino acid. Furthermore, by use of gas chromatography-mass spectroscopy, the sequence and linkages of the components of FL4a were shown to be glueosyl (1-3)fucosyl 1-threonine (2). The determination of the sequence of carbohydrates, the position of the linkage of FIAb, and the anomeric configurations of the carbohydrate linkages of FL3a, FLJb, FCZa, and FL4b are reported here.

RESULTS AND DISCUSSION
Methylation Analysis-The carbohydrate sequence and linkages of FL4b were determined by GC-MS analysis of partially methylated alditol acetate derivatives of the two constituent carbohydrates. A mass chromatogram scan for ion m/z 262 yielded a single peak with a retention time of 5.88 min (Fig. 1A). Likewise, scanning for m/z 264 also revealed a single peak with a retention time of 5.4 min (Fig. 1B). The m/ z 264 is a prominent ion expected for a terminal hexose, and was observed with authentic glucosylpl -+ 3fucosylal --* threonine (Glc-Fuc-Thr) (2). The m/z 262 is a prominent ion expected for 3-linked deoxyhexose and was also previously observed with authentic Glc-Fuc-Thr (2). Mass spectra of the m / z 264 and the m/z 262 peaks were consistent with the presence of a terminal hexose and an internal 3-linked deoxyhexose, respectively (Fig. 2). These mass spectra are comparable to the spectrum of the terminal hexose and 3-deoxyhexose obtained from the PMAA of the carbohydrate residues of authentic Glc-Fuc-Thr (2). Moreover, totally permethylated FL4b yielded essentially the same mass fragments as authentic Glc-Fuc-Thr (figure not shown; see Ref. 2) with the prominent ion being m / z 187 which indicates a terminal hexose (2).
The mass spectra of the PMAA derivatives of FL3a and FL3b were also recorded. Mass chromatograms for ion m / z 234 and m / z 118 revealed a major peak with a time of 4.14 min for both compounds. A mass spectrum of this peak showed ions characteristic for a terminal deoxyhexose (Fig. 3). Enzymatic Degradation of the FL Components-To determine the anomeric configuration of the glucose and fucose residues, the FL components were subjected to treatment with the appropriate glycosidases. Attempts to cleave the terminal glucose of intact FL4a or FL4b with a-or p-glucosidases were not successful. The charge of the amino acid residue could have interfered with the enzymatic cleavage, similar to the experience of Spiro with the glycopeptide glucosyl-galactosyl-hydtoxylsine (9). Therefore, the disaccharide moiety of FL4a and FL4b was cleaved from the amino acid by partial acid hydrolysis and analyzed by gel filtration (Bio-Gel P-2). A single peak which eluted between lactose and raffinose gave an elution pattern similar to that observed for fucosylmannose (10). Furthermore, reduction of the disaccharide followed by acid hydrolysis resulted in quantitative release of the radioactivity as fucitol, whereas acid hydrolysis alone yielded fucose. This result indicates that the fucose moiety is at the reducing end and in turn supports the GC-MS data that the fucose moiety is internal and is likely attached to the amino acid. Moreover, in control experiments, reduction followed by hydrolysis of oligosaccharide chains with nonreducing fucose residues resulted in quantitative release of the radioactivity as fucose. When the disaccharide from FL4a or FL4b was subjected to P-glucosidase treatment, there was quantitative release of the label as fucose (Fig. 4). a-Glucosidase treatment had no effect on the chromatographic mobility of the disaccharide moiety of FL4a or FL4b. These data, in conjunction with the results of methylation linkage analysis, support the interpretation that the glucose moiety of FL4a and FL4b is linked pl-+ 3 to fucose.
The enzymatic determination of the anomeric configuration of the fucose-amino acid linkage in FL4a and FL4b required the prior removal of the terminal glucose residue from both compounds. The removal was accomplished by one round of Smith degradation; FL4a was converted to a compound with the same chromatographic mobility as FL3a (fucosyl-threonine) as seen in Fig. 5. Likewise, FL4b was converted to a compound with the same mobility as FL3b (fucosyl-serine; data not shown). When subjected to a-L-fucosidase treatment, the Smith degradation product of FL4a quantitatively yielded fucose (Fig. 6). The same was true of the Smith degradation product of FL4b (data not shown). Similarly, when authentic FL3a and FL3b were subjected to a-L-fucosidase treatment, fucose was quantitatively released (Fig. 7 ) . Hence, the fucose moieties of FL3a, FL3b, FL4a, and FL4b appear to be a-linked to the amino acid.
Studies have been presented in which four novel amino acid fucosides obtained from rat liver have been further characterized. The proposed structures for the compounds are: FL3a, fucosylal -+ threonine; FL3b, fucosylal -+ serine; F U a , glucosylpl + 3fucosylal -+ threonine; FL4b, glucosyl/31 "+ 3fucosylal -+ serine. FL3a, FL3b, and FL4b have not previously been described in nature. FL4a is likely to be the amino acid fucoside isolated from normal human urine and characterized by Hallgren and co-workers (3). Although the precursor(s) of these components has not been established, preliminary studies from this laboratory have demonstrated that

Characterization of Novel Amino
Acid Fucosides mild base/borohydride treatment, i.e. p-elimination, of a fucose-labeled glycoprotein fraction results in the release of a disaccharide component which contains fucitol and is chromatographically indistinguishable from authentic glucosylpl + 3fucitol (2). Moreover, pulse experiments with [3H]fucose are consistent with the disaccharide-containing glycopeptide being a metabolic precursor of FL4a (2). More recently, it has been found that the disaccharide obtained from the glycoprotein fraction is sensitive to P-glucosidase,4 strongly supporting the idea that the disaccharide is glucosyl-fucose, and in turn is a precursor of the FL4 components. The ,&elimination studies also revealed the release of fucitol from the fucoprotein fraction, thereby suggesting that the fucose is attached to serine or threonine. Hence, it seems reasonable to speculate that the FL components are generated from fucoprotein, and in turn, that the decreased level of FL4a in transformed cells is due to altered metabolism of that protein(s).