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THEJOURNAL OF BIOLOGICAL CHEMISTRY Vol. 256, No. 15, Issue of August 10, pp. 7932-7935, 1981 Printed in U.S.A.
Characterization of Novel Amino AcidFucosides* (Received for publication, February 3, 1981, and in revised form, March 23, 1981)
Martin M. KlingerS, Roger A. Laineg, and Sheldon M.SteinerSI
+
From the School of Biological Sciences and the 8 Department of Biochemistry, The University of Kentucky, Lexington, Kentucky 40506
The structures of FL4band of two otherrelated amino acid fucosides have been determined by a combination of methylationanalysis and enzymatic digestion. Additionally, the anomeric configurations of the carbohydrate moieties of FL4a, previously shownto be glucosyl(1 -+ 3)fucosyl 1 + threonine (Steiner, S., Via, D. P., Klinger, M., Larriba, G., Sramek, S., and Laine,R. (1978) inGlycoproteins and Glycolipids in Disease Processes (Waiborg, E. F., Jr., ed) pp. 378-403, American Chemical Society, Washington,D. C.) have been determined by enzymatic digestion. T h e results indicate that the structures are:FL3a, fucosylal -+ threonine; FL3b, fucosylal + serine; FUa, glucosylpl-+ 3fucosylal+ threonine; andFL4b, glucosylpl-+3fucosylal+ serine. FL4a, which appears to have the same structure as a component from human urine (Hallgren, P., Lundblad, A., and Svensson, S. (1975) J. Biol. Chem. 250, 53125314), and FL4b are highly unusualin that they contain fucose in a nonterminal position. The fucosyl-serine linkage found in compoundsFL3b and FL4b is a novel structure.
Previous studies inthis laboratory have demonstratedthat radioisotopically labeled fucose is incorporated into a seriesof 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 * This investigation was supported by Grant PCM 78152 from the National Science Foundation (to S. M. S.) and Grants GM 23902 and AM 25101 (to R. A. L.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 7 Recipient of Faculty Research Award FRA-131 from the American Cancer Society. ’ The abbreviations used are: NRK, normal rat kidney; MSV-NRK, murine sarcoma virus-transformed NRK; GC-MS, gas chromatography-mass spectroscopy; PMAA, partially methylated alditol acetate derivatives. R. A. Respess, L. S. Kucera, and M. Waite, personal communication.
‘
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 theanomeric configurations of the carbohydrate linkages of FL3a, FLJb, FCZa, and FL4b are reported here. EXPERIMENTALPROCEDURES3 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 of5.88 min (Fig. 1A). Likewise, scanning for m / z 264 also revealed a single peak with a retention timeof 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 spectraof 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 indicatesaterminal 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 characteristicfor a terminaldeoxyhexose (Fig. 3). Portions of this paper (including “Experimental Procedures” and Figs. 4-7) are presented in miniprint at the end of this paper. Miniprint is easily read with the aid of a standard magnifying glass. Full size photocopies are available from the Journal of Biological Chemistry, 9650 Rockville Pike, Bethesda, MD 20014. Request Document No. 81M-246, cite authorb), and include a check for $2.40 per set of photocopies. Full size photocopies are also included in the microfilm edition of the Journal thatis available from Waverly Press.
79132
Characterization of Novel Acid Amino
Scan Number FIG. 1. Mass chromatograms of partially methylated alditol acetate derivativesof the carbohydratemoieties of FUb. The instrumental conditions were: Finnigan 3300-6110 methane-chemical ionization mode:1 torr ion source; pressure sourcetemperature 60 "C; ionizing electron energy, 150 eV, transfer lines, 250 "C. Gas chromatography was performed on a I-m column of 3% OV-210,with the temperature programmed from 140 to 220 "C at a rate of 6 "C/min. The mass chromatogramsof the PMAAs of FL4b were obtained by monitoring for the major ions at m/z 262 ( A ) and m / r 264 ( B )at a rate of one scan every 2 s.
Fucosides
7933
P-2).A single peak which eluted between lactose raffinose and gave an elution pattern similar to thatobserved for fucosylmannose (10). Furthermore,reduction of thedisaccharide followed by acid hydrolysis resulted in quantitativerelease of theradioactivity as fucitol, whereas acidhydrolysis alone the fucose moiety is yielded fucose. This result indicates that a t t h ereducing 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 fromFL4a or FL4b was subjected to P-glucosidase treatment, there was quantitative release of the label as fucose (Fig. 4). a-Glucosidase treatment had effect no on the chromatographic mobility of the disaccharide moiety of FL4a or FL4b. These data, in conjunction with the resultsof 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 removalwas 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 subjecteda-L-fucosidase to 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 tobe a-linked to the aminoacid. 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 notbeen established, preliminary studies from this laboratory have demonstrated that
FIG. 2. Mass spectra of major peaks from mass chromatograms of PMAA's of FUb. A, mass spectrum of majorpeak, retention time 5.88 min, in Fig. 1A. B, mass spectrum of major peak, retention time 5.40 min in Fig. 1B. Enzymatic Degradationof the FL Components-To deterB mine the anomeric configuration of the glucose and fucose 5 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 FIG. 3. Mass spectrum of the major peak, retention time 4.14 moiety of FL4a and FL4b was cleavedfrom the aminoacid by min, found in the analysis of PMAAderivative of FL3a ( A )and partial acid hydrolysis and analyzed by gel filtration (Bio-Gel FL3b ( B ) .
7934
Characterization of Novel Acid Amino
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 thedisaccharide obtained from the glycoprotein fractionis sensitive to P-glucosidase,4strongly supporting the idea that the disaccharide is glucosyl-fucose, and in turn is aprecursor 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 thedecreased level of FL4a in transformed cells is due to altered metabolism of that protein(s). M. M. Klinger and S. M. Steiner, unpublished observations.
Fucosides REFERENCES
1. Via, D. P., Srarnek, S., Larriba, G., and Steiner, S. (1980) J. Cell
B i d 84, 225-234 2. Steiner, S., Via, D. P., Klinger, M., Larriba, G., Sramek, S., and Laine, R. (1978) Glycoproteins and Glycolipids in Disease Processes (Walborg, E.F., Jr., ed) pp. 378-403, American Chemical Society, Washington, D. C. 3. Hallgren, P., Lundblad,A., and Svensson, S. (1975) J . B i d . Chem. 250,5312-5314 4. Larriba, G., Klinger, M., Sramek, S., and Steiner, S. (1977) Biochem. Biophys. Res. Commun. 77, 79-85 5. Skipski, V. P., and Barclay, M.(1969) Methods Enz?mol. 14,530598 6. Trevelyan, W. E., Procter,D. P., and Harrison,J. S. (1950) Nature (Lond.) 166,444-445 7. Spiro, R.G. (1966) Methods Enzymol. 8, 26 8. Hakomori, S. (1964) J. Biochem. (Tokyo) 55, 205-208 9. Spiro, R. G. (1967) J.B i d . Chem. 242,4813-4823 10. Yamada, H., Oshima, Y., Tamura, K., and Miyazaki, T. (1980) Carbohydr. Res.83, 377-378 11. Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951) J. Biol. Chem. 193, 265-275
7935
Characterization of Novel Amino Acid Fucosides SUPPLEMENTARY MATERIAL
to
C h a r a c t e r i z a t i o n o f Novel Amno AcidFucosldes by Martln
H.
K l l n g e r , Roger A . Laine, andSheldon
M. S t e l n e r
EXPERIMENTAL PROCEDURES
Chemicals(Yaltham.
VAS;.).
Preparation o f [ % l f u c o $ e - l a b e l e d FL cmponents NRK c e l l s were labeled"7th [ ~ H I ~ u c o(4.0 s~ u C i l m l 1 f o r 72 hourr. h a r v e s t e d b y r c r a p i n q i n t o 601 ethanol. and processed a s described P r e w o u s l y . The FL'I were scraped and e l u t e d f r o m t h e r i l l c a g e l v l t h Z-pmpanol:NH,OH:H,O 7:Z:l and added t o t h e rat l i v e r e x t m c t a s d e r c n b e d below. P U r l f l C a t l m af t h e FL-CmPOnentsfrom r a t liver. The FL components *ere p u r l f l e d b y a modiflcatlon o f t h e method p r e v i o u s l y d e s c r i b e d (2). B n e f l y , frozen r a t l i v e r s 12.8 kg) were thawed, m x e d w i t h a n equalweight O f c o l d water, Crushed i n d Uarlng blender, and then homogenized i n a B r l n k m n n P D l y t r O n a t r e t t i n g 6 f o r 15 recondr.Ethanol was added t o a f l n a l CDnCFntrationof601,themixture was heated i n a b o 3 l i n g w a t e r b a t h f o r 5 minutes, and then centrifuged. The p e l l e t *as r e e x t r a c t e dw i t h 60% ethanol,thesupernatantspooled,andthen vacuum. The r e s u l t i n g COnCentPate W ~ Ld e l i p i d a t e d by mmxing concentrated byeVaPOratlUnunder W i t h f l v e ~ o l u r n ro f CHC13:CH OH. 2 : ) andshaken v i g o r n u s l y . The upper aqueouI phase was Conc e n t r a t e d and c o h l n e dw i t h[ 3 H ] f u r o r e - l a b e l e dF L ' r 3a 3b 46 and 4b whichhad been prepared fmm NRK c e l l s l a b e l e d i n V i t m * i t h[ 3 H ] f u c o r e .T h l r ' w a r ' c h r d a t a g r a p h e d on d 5 x 85 cm column of SePhadex 6-25 and e l u t e d h 0.2 M p y r i d i n e a c e t a t e pH 5. A l i q u o t so f each f r a c t i o n were counted.andthepeaktuber em Pooled and concentrated by evaporation. This was r t l r r e d w t h d I l u r r y O f AG50x2 ( H I ) . washed w i t hw a t e r and e l u t e dw i t h 0.5N NH OH The e l u a t e was purlf i e df u r t h e r bydescendingpaper chr0matog;aphy i n tw systems: e t t y 1 ' a c e t a t e : a c e t i ca c l d : w a t e r 3.1:l. whichSeparatedFL'S 31 and 3b from 4d and 4b' a f t e r e l u t i o n from thepaper,thelabeled f r a c t i o n s yere run i n e t h y l ~ C e t ~ t e : p y l l d l n e : w d t e r . i 0 . 4 ' 3 . Subsequentchromatography an was r e q u i r e d t o a c h i e v e p u r i t y Of t h eF L ' r a s Judgedby r t a l n i n g rllicagelthinlayerplater w t h a r d n a l l r u l f u r ~ c a c i d and n i n h y d r i n (5). Solvent A , CHC13:CH~Oli:NH40H. 40:80:15. separated FL3a from FL3b and FL4a from FL4b. A l lf o u r compounds were thenchromatographed i nn - b u t a n o l : pyridine:rater,6:4:3; FL'r 3b, 4d and 4b were furtherchmmdtographed in CHC13:CH30H:H20 60.35 0 . Z-prapanal.NHqOH:H20. 7:2:1, and CHClg:CH30H:NHqOH, 1:15:3. r e r p e c t i v e l y .
FIG. 4
Enzyrnatlc degraddtlan
Of
t h ed l r a c c h a r i d em i e t i e lo b t l l n e d
from FL4a and FL4b.
For d e t a l l r o f production o f d i s a c c h a r i d e m i e t l e r and c o n d i t i o n s o f enzyme treatment see E x p t l Proc. A, Hydrolysis product O f disaCCharide frm 13Hl-FL4a f o l l a m g 5 h o w t r e d t m n t w i t h E-glucorldare. B . b o i l e de n l y r eC O n t m fl o pA C, h y d r o l y r i rp r o d u c t Of dildccharide f m m [3H]-FL4b f o l l o w i n g 5 h o u rt r e a t m e n tw i t ht i - g l u c o l l d a l e . 0, b o l l e d enzyme c o n t r o lf o r C. ReactIan mixtures were combined w i t h 0.5 una1 o f unlabeled fucose and chramatographed on Uhdtmn 31*( i n e t h y l a c e t a t e . a c e t i c 1Cid:wdter. 3:l:l f o l 12 h o u r i . The m d t o a c t i w t y vas l o c a t e d b y C u t t i n g 1cm r t n p r f m m O r i g l n t o S o l v e n t f r o n t , a d d i n g 5 0 u l o f Mater t o each s t r i p . and c o u n t i n gi nO m n i f l u o rc o n t a l n l n g 5% Bio-Sal" 885-3. The positionoftheunlabeledstandards was d e r e m l n e d by r t a l n i n g w i t h d l k a l i o e s i l v e r n i t r a t e (6).
I
P T C a r a t i o no fd i s a c c h a ?de from FL4a and FL4b r3HI-FL4a and [3H]-FL4b i s o l a t e d from NRK c e l l s w i t h &ilfucore, *ere h y d m l y z e d w t h 0.2N Hz804 a t lo& f o r 30 minuter The h y d r o l y r a t e r ere d i l u t e d r l t h f o u r v o l u r e ~ o f w a t e r and passed s e q u e n t i a l l y t h r o u g h columnl Of AG50x8 (H+) and AGlz8 ( f o m t e ) f o l l o w e d by e x t e n s i v e washing w i t h wateT. The e l u a t e was conCentrated byeVlPOrdtlOn under vacuum and SUbJeCted t o t h i n l a y e r chromatography i n IOlYent A. The disaccharide reglon was scraped and e l u t e d i n CH$H:HZO, 1:l ( u l v ) .
M *
3
9"
FIG 5 S m t hd e q r a d a t i o n O f []HI FL4a. A, u n t r e a t e d[ % I FL3a standard. B. u n t r e a t e d ['HI -standard. C. I3H1 FL4a f o l l m l n q one round O f Smithdegradation. 0. 13Hl FL4a f o l l o * l n gm i l da c i dh y d r a l y s i lw i t h o u tp r i o rp e r t o d a t et r e a t m e n t . Samples were analyzedby thinlayerchromatography i n solvent A. C o n d i t i o n so fp e r i o d a t et r e a t r e n t , reduction. and mmld a c i d h y d r o l y s i s a r e d e t a i l e d i n E x p t l . Pmc.
PrOtelOdeterininatlonProtein
was w a n t 7 f i e d by t h e method o f L m r y e t d l .
(11)
M e t h y l a t i o na n a l y s i sP a r t l l l l ym e t h y l a t e dd l d l t o l a c e t a t e d e l l l v a t i v e s O f thecarbohydrate m o i e t i e s O f the FL'r from r a t l i v e r were prepared a$ follows. Sampler(60-100nmol)of each of and sonicatedfor 15 seconds. Methylt h eF L ' r were Suspended i nd i m e t h y lS u l f o x i d e( 0 . 2 ~ 1 ) IUlflnylmthylCdrbanion(0.2ml)preparedbythe method O f HakOmOrl (8) was addedunder m t r o g e n and t h em i x t u r eS o n i c a t e df o r one hour.Methyliodlde ( 0 . 5 m l ) was added d m p y l w u n d e rn i t r o g e n and t h e m1XtUfe s o n i c a t e df o r one hour. A f t e r removal of excess methylIodide by b r i e f evapor a t i o nu n d e r a stream O f nitrogen,Chloroform(Zml) and water (2.1) were added. The SOlut10n *ai shahen vigorously. centrifuged. and the aqueous upper phase discarded. The l a e r phase was additlan r e e x t r a c t e d f o u r more times r i t h w a t e r (3.1) andthen d r i e d u n d e r n i t r o g e n a f t e r t h e O f ethanol (0 5ml). The samples were t h e nm t h a n o l y z e di n 1N methanolic HCI (0.5ml) a t l0OC f o r 3 hOUT'I, cooled, and d r i e d undeP nitrogen.Freshlyprepared ZN t l i f l U o m a C e t i Ca c l d( 0 . 2 5 m l ) *as added and the 1dmD1el incubated a t lZ0C f o r one h o u rA f t e rt h em x t u r e s were Cooled and evaporatedundernitrogen. 0.2M Sodium borodeuterlde i n 1M HHqOH(O.5 m l ) was added t o each tube and t h e ~ d l w l e s were reduced I t mom temperatureOvernight.Concentratedacetic acid was added d r a p w l r et od e s t r o y excess Na004 and themixtures were thenevaporatedrepeatedly vlth methanol. The d r l e dr a m p l e i yew resuspended i np y l i d i n e ( 0 . 2 5 m l ) and l c e t l ca n h y d r i d e ( 0 25 m l ) ,s o n i c a t e df o r 15 IecOndS, and heated a t lO0C f o r one houT. The mixtures were p a r t i t i o n e d l l t h c h l o r o f o r m (2ml) and ~ a t e r ( 3 m l ) , r e n t r l f u g e d , t h e u p p e r aqueous phasediscarded, and the lower phase r e r t r a c t e dw i t hw a t e v The d r i e d lorer phase *as resuspended i n lOul O f acetone conditions of gas and 1-2 "1 was used d l r e c t l y f o r gas Chrmdtography-massspectrometry.For Chromatography-massSpectroscopy, see legend t o F i g 1.
cy F R Y c s I u *
a - L - f u C o s i d d L et r e a t r e n to f Smilth degraded[3nl-FL4a. A, Smithdegraded 8 . b o i l e d enzyme c o n t r o l . FL4a f o l l o w i n g 4 day i n c u b a t i o n p w l o d Witha-L-fUCOsldale. see F i g 4. F o rc o n d i t i o n so f enzyme t r e a t m o t , see Forchromatographyconditionr, o f free fucose E r p t l . Pmc. A r r ai n d i c a t e sm r g r a t i o n
