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Presence of Vicia graminea lectin- or Vicia unijuga lectin-binding (Vgu) glycoproteins, Vgu glycoproteins with Thomsen-Friedenreich (T) activity and T-active glycoproteins in human meconium
ht.
Pergamon
J. &a-hem.
Cell Bid. Vol. 27, No. 3, pp. 319-327,
1995 Copyright @J 199s Elsevier Sciice Ltd Printed in Great Britain. All rightsreserved 13S7-2725/95
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Presence of Vicia graminea Lectin- or Vicia (vgu) Glycoproteins, vgu Led wekh (T) Activity Glyeqwote~ns with Thomsen-F and T-active Glycoproteins in Human Meconium NOBORU UCHIDE,” KUNIO OHYAMA,? TOSHIO YAMAKAWA, SEIICHI OHKUMA Department of Biochemistry, Tokyo 192-03, Japan
Tokyo College of Pharmacy, 1432- 1 Horinouchi, Hachioji,
Perchioric acid&uble fraction prepared from a mixture of meco~‘urnsof 22 newborn babies was sub+ted to a systematic aflinity chromatography using Vi& n@nga led&Ccolumn and Aruchis hypoguea lectin-Agarose column and aquated iato four fmctkms, Vgu glycupmtein, Vgu giycoprotein with T activity, T-active glycoprotein and another glyqmteia fractions. HPLC anaiysisshowedthat the first fraction contained three glveopnrteiirs with n&a&r weight (M,) of about 19,4@0kDa, 55OOkDa and BOOkDa, the second fraction co&&d of two glycoproteins with M,, of about 460 kDa and 150 kDa and the third fraction composed of several glycoproteins including the main glycoprotein with M, of about 11,700 kDa. Keywords: Vgu glycoprotein oncofetal antigen
Vicia graminea
lectin
Viciu unij8gu lectin
human meconium
Int. .l. Biochem. Cell Biol. (1995) 27, 319-327
T activity have been isolated, in a high state of purity, as mucin-type glycoproteins (Feizi et al., 1984) with very high molecular weights from cyst fluids of human ovarian clear cell carcinoma (Yanagi et al., 199Ob), a&tic fluids of primary ovarian cancer patients (Hayashi et al., 1990) and human liver metastases from pancreas carcinoma (Ueno et al., 1992) and breast carcinoma (Ishigure et al., 199 1) either by a systematic affinity chromatography (Yanagi et al., 1990b; Hayashi et al., 1990; Ishigure et al., 1991) using VUA and PNA as immobilized ligands or by the combined use of the systematic affinity chromatography and high performance liquid chromatography (HPLC) using Asahipak GS-7 10 column (Ueno et al., 1992). Before then, Springer et al. (1966) had separated a VGAreactive and MN-inactive glycoprotein from human meconium by a conventional separation technique and designated its glycoprotein meconium-Vg antigen. More recently, &hide et al. (1993) have found that all of perchloric
INTRODUCTION
Recently, Ohkuma and Yanagi (1992) have elucidated that Vicia graminea lectin (VGA) and Vicia unijuga lectin (VUA) selectively recognize a small glyco (sialoglyco)-oligopeptide moiety (A) or (B) in glycoprotein molecules, respectively (Fig. I), and proposed that glycoproteins, which react with VGA and VUA, but do not react with anti-M and anti-N sera, are designated as a whole VGA- or VUA-binding (Vgu) glycoproteins, and Vgu glycoproteins, which also react with Arachis hypogaea lectin [PNA, anti-T (Thomsen-Friedenreich) lectin] and/or anti-T serum, are named Vgu glycoproteins with T activity. On the one hand, by then, a series of Vgu glycoproteins and Vgu glycoproteins with *Present address: Molecular Pharmacology Group, Tsukuba Research Laboratories, Nippon Glaxo Co., Ltd, 43 Wadai, Tsukuba, Ibaraki 300-43, Japan. tTo whom all correspondence should be addressed. (Received I8 January 1994; accepted 7 November 1994). 319
Noboru Uchide et al.
320
3
6
prepared from V. unijuga leaves by the method of Yanagi et al. (1990a). Influenza A and B viruses used in this work were PR8 strain and Osaka strain, respectively. Calibration reference substances were products of Sigma Chemical Co. (St Louis, MO., U.S.A.). VUA-Cellulofine was prepared by the method of Yanagi et al. (1990b). PNA-Agarose was obtained from Pharmacia Fine Chemical AB (Uppsala, Sweden). Asahipak GS-710 (vinyl alcohol polymer, different molecular weight range lo-lo4 kDa) column was a product of Asahi Chemical Industry Co., Ltd (Kawasaki, Kanagawa, Japan).
t
t
Chemical composition analysis
-Ser-Thr-Thr-
T:
Ts:
GalPl
----)
Gala1 4
-
Ser -Thr-
3GalNAca
+
1 -0
-
JGalNAca
---)
1 -0
-
NeuAcai NeuAcai Fig. 1. Small glyco (sialoglyco)-oligopeptide moiety (A) or (B) in glyoprotein molecules.
acid-soluble fractions (PASFs) from human meconiums reacted with VGA and VUA and very weakly reacted with both the T reagents, but did not react with anti-M and -N set-a.Therefore, from the studies of Springer et al. (1966) and Uchide et al. (1993) and the findings of Ohkuma and Yanagi (1992), it is possible to infer that human meconium contains Vgu glycoproteins and/or Vgu glycoproteins with T activity. To confirm the presence of Vgu glycoproteins and/or Vgu glycoproteins with T activity in human meconium, separation and characterization of these glycoproteins from human meconium by the systematic affinity chromatography, were studied in this work. MATERIALS
AND METHODS
Materials
PASF prepared from a mixture of meconiums of 22 newborn babies (blood-group type AMN, BMN, ABMN or OMN) by the perchloric acid method (Krupey et al., 1972) in the preceding study (Uchide et al., 1993), was used in this work. Anti-A, -B, -M and -N sera were purchased from Ortho Diagnostic System Inc. (Norcross, Ga, U.S.A.). Anti-H serum was prepared from Japanese eels (Angilla juponica) by the method of Yamamoto and Takizawa (I 98 1). Anti-T serum was prepared by immunizing rabbits with neuraminidase-treated human erythrocytes (OMN) according to the method of ijrntoft et al. (1985). VGA, PNA, Canavalia ensiformis lectin (Con A) and Phaseolus vulgaris lectin (PHA-E) were purchased from E-Y laboratories (San Mateo, Calif. U.S.A.). VUA was
Sialic acid, neutral sugar, amino sugars and amino acids were determined according to the methods used by Ohyama et al. (1993). HPLC
HPLC was carried out by using Rhodyne sample injector 7 125, Shimadzu constametric pumps LC-6A, Shimadzu photodiode array u.v.vis detector SPD-M6A and Asahipak GS-710 column (0.76 x 50 cm) under the conditions of a flow rate of 0.5 ml/min with 0.2 M phosphate buffer (pH 6.0) as an elution buffer. A 0.5 mg sample of V, VP or P fraction was dissolved in 50,ul of the same buffer and applied to the column. Eluates (each 0.25 ml) were collected and absorbance at 206 nm and reactivities for VUA and/or PNA of each of the eluates by ELISA were estimated. Apparent molecular weight (M,) estimation
M,s of Vl-V3, VPl, VP2 and Pm were estimated by HPLC. The relation between log molecular weight (Y, Da) of a sample (glycoprotein or reference substance) and its retention time (X, min) in HPLC was shown by the following equation: Y = -0.031071X2
+ 1.4127X -8.7525
This equation was calculated with Macintosh IIci computer (Apple Computer Inc., Cupertino, Calif., U.S.A.) using a soft program, CA-Cricket’“’ Graph IIITM (Computer Associates International Inc., U.S.A.). Ferritin (M, 450 kDa. Retention time (Rt) was 30.1 (min) in HPLC), apoferritin (M, 443 kDa, Rt 30.1) /3-amylase (M, 200 kDa, Rt 30.7) aldolase (M, 158 kDa, Rt 31.0) human IgG (M, 150 kDa, Rt 30.7) alcohol dehydrogenase (M, 150 kDa, Rt 31.4), ovalbumin (M, 45 kDa, Rt 31.8) and chymotrypsinogen (M, 25 kDa, Rt 32.4) were
Glycoproteins
used as calibration apparent molecular HPLC.
in human
reference substances in weight estimation by
Hemagglutination inhibitory assay Hemagglutination inhibitory assay of PASF and V, VP, P, Ul and U2 fractions was carried out by the method of Yanagi et al. (1990b). Enzyme-linked immunosorbent assay (ELBA) ELISA was carried out according to the method of Ueno et al. (1992). Systematic aJinity chromatography using VUAcellulojine column and PNA -agarose column of PASF In affinity chromatography, VUA-Cellulofine and PNA-Agarose columns previously equilibrated with buffer A (0.1 M Tris-HCl buffer (pH 7.2) containing 0.3 M NaCl) at 3-4°C were used and absorbance at 280 nm and reactivities for VUA and/or PNA of the effluent and eluate were estimated. Affinity chromatography and dialysis of the pooled effluents or eluates against distilled water were performed at 3-4°C. In affinity chromatography using VUA-Cellulofine column, the efflux and elution were carried out at in a flow rate of 3 ml/hr and the effluents or eluates (each 3 ml) were collected. A 30.0 mg sample of PASF was dissolved in 3.0 ml of buffer A and applied to a column (1 .O x 8.0 cm) of VUA-Cellulofine. The column was washed with buffer A and the effluents were pooled, dialyzed and lyophilized. The dry residue was
321
meconium
designated VUA-unbound fraction (Fig. 2). Bound matters on the column were eluted with 3.0 M KSCN in buffer A. The eluates were pooled, dialyzed and lyophilized. The dry residue was named VUA-bound fraction (Fig. 2). In affinity chromatography using PNA-Agarose column, the efflux and elution were all performed in a flow rate of 1 ml/hr and the effluents or eluates (each 1 ml) were collected. A 2.0 mg sample of VUA-bound fraction was dissolved in 1 ml of buffer A and applied to a column (1 .l x 2.0 cm) of PNA-Agarose. The column was washed with buffer A and the effluents were pooled, dialyzed and lyophilized. The dry residue was named VUA-bound and PNA-unbound (V) fraction (Fig. 3). Bound matters on the column were eluted with 0.2 M lactose in buffer A. The eluates were pooled, dialyzed and lyophilized. The dry residue was designated VUA-bound and PNA-bound (VP) fraction (Fig. 3). A 2.0 mg sample of VPunbound fraction was dissolved in 1 ml of buffer A and applied to a column (1.1 x 3.0 cm) of PNA-Agarose. The column was washed with buffer A and the effluents were pooled, dialyzed and lyophilized. The dry residue of two fractions were named VUA-unbound and PNA-unbound 1 (Ul) and 2 (U2) fractions, respectively (Fig. 4). Bound matters on the column were eluted with 0.2 M lactose in buffer A. The eluates were pooled, dialyzed and lyophilized. The dry residue was designated VUA-unbound and PNA-bound (P) fraction (Fig. 4).
VUA-bound fraction t I
0.0
0.0 10
0
20
30
40
Tube number Fig. 2. Affinity
chromatography
using
VUA-Cellulofine
column
of PASF
322
Noboru Uchide et al. VUA-bound and PNA-unboud l a2 (V) fraction
VUA-bound and PNA-bound (VP) fraction
I
I
0.9
nc lJ.0
0.3
0.0
0
10
20
: 0
30
Tube number Fig. 3. Affinity chromatography
using PNA-Agarose
RESULTS
Separation of the fractions of Vgu glycoprotein (V), Vgu glycoprotein with T activity (VP), T active glycoprotein (P) and other glycoproteins (171and U2) from PASF by a systematic afinity chromatography using VUA-Cellulojne column and PNA -Agarose column
PASF, which was prepared from a mixture of meconiums of 22 newborn babies (AMN, BMN, ABMN or OMN) was systematically subjected to affinity chromatography using
10
fraction.
VUA-Cellulofine column and PNA-Agarose column, as shown in Figs 2-4. In the first affinity chromatography using VUA-Cellulofine column, PASF into VUAwas separated unbound fraction and VUA-bound fraction (Fig. 2). In the second affinity chromatography using PNA-Agarose column, VUA-bound fraction was senarated into VUA-bound and PNAunbound &) fraction and VUA-bound and PNA-bound (VP) fraction (Fig. 3). In the third affinity chromatography using PNA-Agarose column, VUA-unbound fractions were separated
VUA-unbound and PNA-unbound 2 (U2) fraction
VUA-unbound and PNA-unbound 1 (Ul) fraction
0
column of VUA-bound
VUA-unbound and
20
L
,O
30
Tube number Fig. 4. Affinity chromatography
using PNA-Agarose
column of VUA-unbound
fraction.
Glycoproteins in human meconium
into VUA-unbound and PNA-unbound 1 (Ul) and 2 (U2) fractions and VUA-unbound and PNA-bound (P) fraction (Fig. 4). The fractions of V, VP, P, UI and U2 were obtained as a white solid, respectively. Yields of these fractions shown in Table 1 indicate that U2 fraction is the main fraction of PASF, P fraction is the second main fraction and each of the fractions of V and VP is the miner fraction. Chemical compositions and serological properties of the fractions of V, VP, P, UI and U2
All the fractions of V, VP, P, Ul and U2 were demonstrated to be glycoproteins containing sialic acid by their chemical composition analysis, as shown in Table 1. The notable features of the carbohydrate compositions of the five fractions were that these fractions contained a large amount of N-acetylgalactosamine, respectively, P fraction did not contain mannose and V, Ul and U2 fractions contained a very small amount of
323
mannose, respectively. The total contents of threonine and serine were considerably higher than that of aspartic acid in the five fractions. The reactivities of the five fractions with 13 hemagglutinins are presented in Table 2. All the fractions reacted with anti-A and -B sera, but did not react with anti-M and -N sera. Among these fractions, only Ul and U2 fractions reacted with anti-H serum. V fraction gave positive reactions with VGA and VUA and showed negative reactions with anti-T serum and PNA. VP fraction gave positive reactions with VGA, VUA and PNA and showed a very weak reaction with anti-T serum. V and VP fractions showed essentially the same reactivities with ConA, PHA-E and influenza viruses. P fraction gave a positive reaction with PNA, showed a very weak reaction with anti-T serum and gave negative reactions with VGA, VUA, ConA, PHA-E and influenza viruses. Ul and U2 fractions did not react with VGA and VUA and
Table 1. Yields and chemical compositions of V, VP, P, Ul and U2 fractions separated from PASF by a systematic affinity chromatography using VUA-Cellulofine column and PNA-Agarose column
Fraction
PASF”
VUA-bound and PNA-unbound (V)
Yield (mg/lOO mg of PASF)
151.9b
0.2
Carbohydrate composition (w/w,%) Sialic acid 9.3 Glucose n.d. Mannose 4.8 Galactose 22.8 Fucose 8.3 N-Acetylglucosamine 19.2 N-Acetylgalactosamine 9.1
2.8 1.2 0.8 8.4 2.8 27.8 19.7
Amino acid composition (mol/lOO mol) 10.4 7.5 Asp Thr 17.6 7.6 Ser 11.7 19.1 Glu 10.0 9.8 Pro 9.5 4.4 8.0 17.3 GUY .41a 7.8 8.9 1.1 n.d. CYS Val 6.1 4.6 Met 1.3 1.3 lie 3.4 2.5 Leu 7.6 4.2 1.7 1.7 Tyr Phe 3.7 2.3 4.6 4.1 LYS His 2.8 2.8 2.4 1.8 Arg nd., not detected. “cited from the paper of Uchide et al. (1993). bmg/g of wet human meconium.
VUA-bound and PNA-bound (VP)
VUA-unbound and PNA-bound (P)
0.2
19.0
VUA-unbound VUA-unbound and and PNA-unbound I PNA-unbound 2 (LJ2) u-J11 13.2
7.0
3.3
8.1
nd.
nd. n.d.
nd.
4.6 17.2 7.1 11.8 12.3 4.2 24.8 16.0 4.4 10.8
28.2 4.4 22.9 16.4
1.6 21.3 8.9 18.7 11.9
5.5
9.7
4.5 25.4 17.7 4.8 11.6 6.5 8.3
nd.
nd.
4.5 0.2 2.7 4.2
4.8
5.5
I.0
1.9 3.3
10.1
1.1 1.6 1.8 1.7 2.3
2.4 3.6 0.8 1.3 2.2 2.5 2.7
24.4 11.9 7.6 10.3 8.3 6.1 0.7
5.1 1.5 2.3 2.3 1.6 1.8
54.0
7.0 0.2 1.4
5.9 2.8 18.2
16.4 IO." 11.1 13.0 Il.’ 1.7 I I.2 ?i 0.4 4.4
I.3 27 6.3
17 31 41
12 2s
Noboru Uchide et al.
324
Table 2. Hemagglutination inhibition activities of V, VP, P, UI and U2 fractions separated from PASF by a systematic affinity chromatography using VUA-Cellulofine column and PNA-Agarose column
Fraction
PASF”
Hemagglutinin
(titer = 8)
Anti-A serum Anti-B serum Anti-H eel serum Anti-M serum Anti-N serum Anti-T serum Arachis hypogaea (PNA, anti-T lectin) Vi& gruminea (VGA) Vicia unijugu (VUA) Canavalia
1 2 1250
n.i. n.i. v.w.r. v.w.r.
VUA-bound VUA-bound VUA-unbound VUA-unbound VUA-unbound and and and and and PNA-unbound PNA-bound PNA-bound PNA-unbound 1 PNA-unbound 2 (V (VP) m W1) (W Minimum concentration @g/ml) of fraction giving complete inhibition 156
10
78
20
n.i. n.i. n.i. n.i. n.i.
n.i. n.i. n.i. V.W.T.
n.i. n.i. n.i. v.w.r.
39
313 78
20
39 39
1 10
5 5 313
1250
156
xi. n.i. v.w.r. v.w.r.
n.i. n.i. v.w.r. v.w.r.
n.i. n.i. n.i.
n.i. n.i. 1%
n.i. xi. 1250
ensiformis
5000 1250 1250
n.i.
10 n.i.
vulgaris
1250
2500
1250
n.i.
625
n.i.
313 625
625 2500
1250 1250
n.i. n.i.
39 39
1250 1250
(Con A) Phaseolus
(PHA-E) Influenza A virus Influenza B virus
n.i., no inhibition of hemagglutination at 5000 pg/ml. v.w.r., a very weak reaction. “cited from the paper of Uchide et al. (1993)
gave very weak reactions with both the anti-T reagents, while reacted with ConA and Influenza viruses. HPLC analyses of the fractions of V, VP and P In HPLC, V fraction gave numerous VUA-reactive peaks including the three main peaks, VI, V2 and V3, as shown in Fig. 5. The elution positions of VUA-reactive peaks did not agree with those of the peaks of absorbance at 206 nm. Apparent M,s of
19,364 kDa, 5491 kDa and 1871 kDa were found for VI, V2 and V3 by HPLC, respectively. VP fraction gave VP1 and VP2 peaks in HPLC, as shown in Fig. 6. The elution positions of VUA-reactive peaks agreed substantially with those of PNA-reactive peaks, but differed from those of the peaks of absorbance at 206 nm. Apparent M,s of 462 kDa and 152 kDa were found for VP1 and VP2 by HPLC, respectively. As seen in Fig. 7, P fraction gave the main PNA-reactive peak, Pm, with M, of
Tube number Fig. 5. HPLC analysis of V fraction
Glycoproteins in human meconium
325
Tube number Fig. 6. HPLC analysis of VP fraction
11,680 kDa, in HPLC. The elution position of Pm differed from those of peaks of absorbance at 206nm. DISCUSSION
In the preceding paper (Uchide et al. 1993), we have reported that PASF extracted from a mixture of meconiums of 22 newborn babies with blood group AMN, BMN, ABMN or OMN type by the perchloric acid method (Krupey et al., 1972), reacted with VGA and VUA and also reacted very weakly with PNA and anti-T serum, but did not react with anti-M
and -N sera. In this work, to clarify the presence of Vgu glycoproteins and/or Vgu glycoproteins with T activity in human meconium, PASF was subjected to a systematic affinity chromatography using VUA and PNA as immobilized ligands. First, PASF was separated into VUAunbound fraction and VUA-bound fraction in affinity chromatography with VUA-Cellulofine column (Fig. 2). Secondly, VUA-bound fraction was separated into VUA-bound and PNAunbound (V) fraction and VUA-bound and PNA-bound (VP) fraction in affinity chromatography with PNA-Agarose column (Fig. 3). Thirdly, VUA-unbound fraction was separated
Pm
Tube number Fig. 7. HPLC analysis of P fraction
326
Noboru Uchide et al.
into VUA-unbound and PNA-unbound I (Ul) VUA-reactive and MN-inactive glycoproteins and 2 (U2) fractions and VUA-unbound and have been designated Vgu glycoproteins and PNA-bound (P) fraction in affinity chromaVgu glycoproteins, which also react with PNA tography with PNA-Agarose column (Fig. 4). and/or anti-T serum, have been named Vgu The five fractions, V, VP, P, Ul and U2, glycoproteins with T activity (Ohkuma and were sugar rich glycoprotein fractions with Yanagi, 1992). Accordingly, it is apparent that V and VP fractions are a Vgu glycoprotein 60.0, 63.5, 75.2, 70.5 and 52.4% carbohydrate, respectively. V fraction contained N-acetylfraction and a fraction of Vgu glycoprotein with galactosamine and serine in high percentages, T activity, respectively, and P fraction is a mannose in a very low percentage and aspartic T-active glycoprotein fraction, while Ul and U2 acid in a low percentage (Table 1). The fractions are a glycoprotein fraction without N-acetylgalactosamine content was much having Vgu and T activities, respectively, and higher than the mannose content in V and VP that human meconium contains Vgu glycofractions which contained aspartic acid in a low proteins, Vgu glycoproteins with T activity and percentage, respectively. P fraction contained T-active glycoproteins. N-acetylgalactosamine and serine in high perTo confirm the glycoprotein composition of centages and threonine in a very high percent- V, VP, and P fractions, these fractions were age, but did not contain mannose (Table 1). analyzed by HPLC (Figs 5-7). The analysis It is well known that the N-acetylgalactosamine results indicate that V fractions contained three residue is a characteristic component of O-gly- Vgu glycoproteins, Vl glycoprotein with M, of cosidically linked saccharide chain (mucin-type about 19,400 kDa, V2 glycoprotein with M, sugar chain) and the serine and threonine of 5500 kDa and V3 Glycoprotein with M, of residues are essential for the formation of O-gly1900 kDa, VP fraction consisted of two kinds cosidic linkage, on the one hand, the mannose of Vgu glycoprotein with T activity, VP1 glycoresidue is characteristically contained in the protein with M, of about 460 kDa and VP2 N-glycosidically linked saccharide chain (as- glycoprotein with M, of 150 kDa and P fraction paragine-linked sugar chain) and the asparagine composed of several T-active glycoproteins (aspartic acid) residue is the essential amino acid including the main glycoprotein (Pm glycoprotein) with M, of about 11,700 kDa. VI-V3, component of N-glycosidic linkage (Kobata, VPl, VP2 and Pm glycoproteins seem to be 1984). Consequently, the carbohydrate composition analyses of the fractions of V, VP and P a kind of mucin-type glycoproteins (Feizi et al., seem to show that the mucin-type sugar chains 1984), because these glycoproteins bear high are more remarkably abundant than the as- molecular weight and abundant mucin-type paragine-linked sugar chains in V and VP frac- sugar chains, respectively, as described above. tions and, on the other hand, P fraction Proteins or antigens, which occur in common abundantly contains only the mucin-type sugar both in carcinoma tissues and fetus tissues, were chains. termed carcinofetal proteins (Nishi, 198 l), All of PASF and its five fractions, V, VP, P, oncofetal proteins (Yachi, 1981) or oncofetal Ul and U2, showed no reactions with anti-M antigens (Waldman and Herberman, 1982). On and -N sera. V and VP fractions reacted with the one hand, before then, meconium-Vg glycoVGA and VUA, and PASF reacted weakly with protein had been separated from human the same two lectins, but P, Ul and U2 fractions meconium (Springer et al., 1966) and then did not react with the same two lectins. VP and Vl-V3 glycoproteins (Vgu glycoproteins), VP1 P fractions reacted with PNA, and PASF and and VP2 glycoproteins (Vgu glycoproteins with the fractions of U 1 and U2 reacted very weakly T activity) and Pm glycoprotein (T-active glycowith PNA, but V fraction did not react with the protein) were isolated from human meconium same lectin. V fraction did not react with anti-T in this work. On the other hand, it has been serum, and PASF and the fractions of VP, P, U 1 found that all 21 kinds of PASFs from human and U2 reacted very weakly with the same intact tissues (the brain, submaxillary gland, anti-serum (Table 2). Therefore, the serological esophagus, stomach, small intestine, large study described here seems to show that Vgu intestine, liver, gallbladder, pancreas, lung, activity of PASF was concentrated in its V and heart, spleen, thymus gland, kidney, bladder, VP fractions and T activity of PASF was adrenal gland, prostatic gland, ovary, skeletal concentrated in its VP and P fractions. As muscle and skin) and human normal serum described in introduction, recently, VGA- or gave no reactions with VGA, VUA, PNA and
Glycoproteins in human meconium
anti-M and -N sera (Ohyama et al., 1989) and reported that a series of Vgu glycoproteins, Vgu glycoproteins with T activity and T-active glycoproteins were isolated from human liver metastases of pancreas carcinoma (Ueno et a[., 1992) and breast carcinoma (Ishigure et al., 1991), cyst fluids of human ovarian clear cell carcinoma (Yanagi et al., 1990b) and ascitic fluids of primary ovarian cancer patients (Hayashi et al., 1990) and also detected in human primary hepatocellular carcinoma (Otsuka et al., 1992) human liver metastases of sigmoid colon carcinoma (Otsuka et al., 1988, 1991a) and ascitic fluids of liver cancer patients (Otsuka et al., 1991b). Therefore, it may be concluded that Vgu glycoproteins, Vgu glycoproteins with T activity, T-active glycoproteins and meconium-Vg glycoprotein are new carcinofetal glycoproteins, oncofetal glycoproteins or oncofetal antigens, respectively. REFERENCES
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glycophorins B, C and D from human erythrocyte membranes. Biochim. Biophys. Acta 1148, 133-138. C)mtoft T., Mors N. P. O., Eriksen G., Jacobsen N. J. and Paulsen H. S. (1985) Comparative immunoperoxidase demonstration of T-antigen in human colorectal carcinoma and morphologically abnormal mucosa. Cancer Res. 45, 447-452.
Otsuka K., Ohkuma S., Nakajima T. and Kudo T. (1988) Thomsen-Friedenreich (T)-active glycoproteins and a blood group N antigen precursor with T activity from human liver metastatic carcinomas. Naturwissenschuften 75, 407-409. Otsuka K., Kudo T., Nakajima T. and Ohkuma S. (1991a) Blood group N antigen precursor glycoproteins and N antigen precursor glycoproteins with ThomsenFriedenreich (T) activity from human liver metastatic carcinomas. In!. J. Biochem. 23, 569-578. Otsuka K., Nishida S., Yuasa H., Hara H., Shirai T. and Ohkuma S. (199lb) Thomsen-Friedenreich (T)-active glycoproteins, blood group N antigen precursor glycoproteins and N antigen precursor glycoproteins with T activity from ascitic fluids of liver cancer patients. Int. J. Biochem.
23, 827-837.
Otsuka K., Mizuno M., Kagawa K., Kashima K. and Ohkuma S. (1992) Presence of a Vi& unijuga lectinbinding (Vgu) glycoprotein with Thomsen-Friedenreich (T) activity and Vgu glycoproteins in human primary hepatocellular carcinoma. Int. J. Biochem. 24, 1407-1419. Springer G. F., Nagai Y. and Tegtmeyer H. (1966) Isolation and properties of human blood-group NN and meconium-Vg antigens. Biochemistry 5, 3254-3272. Uchide N., Ohyama K., Yamakawa T., Watanabe H., Hirakawa S., Yoneyama K. and Ohkuma S. (1993) Serological property of perchloric acid-soluble glycoprotein fractions of human meconiums. Yakugaku Zasshi (J. pharm.
Sot. Jpn)
113, 532-536.
Ueno E., Otsuka K., Kudo T. and Ohkuma S. (1992) Isolation and characterization of a Viciu gramineu and Vicia unijugu lectins-binding (Vgu) glycoprotein with Thomsen-Friedenreich (T) activity from human liver metastases of pancreas carcinoma. Inr. J. B&hem. 24, 769-776. Waldman T. A. and Herberman R. B. (1982) Tumor markers in diagnosis and monitoring therapy. In Cancer Medicine (Edited by Holland J. F. and Frei E.), Vol. XV-2, III pp. 1069-1089. Lea & Febiger, Philadelphia. Yachi A. (1981) Isoferritin and other oncofetal proteins. Taisha
(Merab.
Dis.),
18, 731-740.
Yamamoto S. and Takizawa H. (1981) Serological studies on biological components of fishes and shellfishes. Screening of so-called anti-H agglutinin and anti-H precipitin contained in eel sera and their rapid mutual separation. Rep. natn.
Res. Inst.
Police
Sci. 34, 205-213.
Yanagi K., Ohyama K., Yamakawa T., Hashimoto K. and Ohkuma S. (1990a) Purification and characterization of anti-N lectin from Vicia unijuga leaves. Inr. J. Riochem. 22, 43-52. Yanagi K., Ohyama K., Yamakawa T. and Ohkuma, S. (1990b) Isolation and characterization of blood group N antigen precursor glycoproteins with ThomsenFriedenreich (T) activity, N antigen precursor glycoproteins and T-active glycoproteins from cyst fluids of malignant ovarian clear cell carcinoma. Inf. J. Biochem. 22, 1015-1028.
Pergamon
J. &a-hem.
Cell Bid. Vol. 27, No. 3, pp. 319-327,
1995 Copyright @J 199s Elsevier Sciice Ltd Printed in Great Britain. All rightsreserved 13S7-2725/95
$9.50 + 0.00
Presence of Vicia graminea Lectin- or Vicia (vgu) Glycoproteins, vgu Led wekh (T) Activity Glyeqwote~ns with Thomsen-F and T-active Glycoproteins in Human Meconium NOBORU UCHIDE,” KUNIO OHYAMA,? TOSHIO YAMAKAWA, SEIICHI OHKUMA Department of Biochemistry, Tokyo 192-03, Japan
Tokyo College of Pharmacy, 1432- 1 Horinouchi, Hachioji,
Perchioric acid&uble fraction prepared from a mixture of meco~‘urnsof 22 newborn babies was sub+ted to a systematic aflinity chromatography using Vi& n@nga led&Ccolumn and Aruchis hypoguea lectin-Agarose column and aquated iato four fmctkms, Vgu glycupmtein, Vgu giycoprotein with T activity, T-active glycoprotein and another glyqmteia fractions. HPLC anaiysisshowedthat the first fraction contained three glveopnrteiirs with n&a&r weight (M,) of about 19,4@0kDa, 55OOkDa and BOOkDa, the second fraction co&&d of two glycoproteins with M,, of about 460 kDa and 150 kDa and the third fraction composed of several glycoproteins including the main glycoprotein with M, of about 11,700 kDa. Keywords: Vgu glycoprotein oncofetal antigen
Vicia graminea
lectin
Viciu unij8gu lectin
human meconium
Int. .l. Biochem. Cell Biol. (1995) 27, 319-327
T activity have been isolated, in a high state of purity, as mucin-type glycoproteins (Feizi et al., 1984) with very high molecular weights from cyst fluids of human ovarian clear cell carcinoma (Yanagi et al., 199Ob), a&tic fluids of primary ovarian cancer patients (Hayashi et al., 1990) and human liver metastases from pancreas carcinoma (Ueno et al., 1992) and breast carcinoma (Ishigure et al., 199 1) either by a systematic affinity chromatography (Yanagi et al., 1990b; Hayashi et al., 1990; Ishigure et al., 1991) using VUA and PNA as immobilized ligands or by the combined use of the systematic affinity chromatography and high performance liquid chromatography (HPLC) using Asahipak GS-7 10 column (Ueno et al., 1992). Before then, Springer et al. (1966) had separated a VGAreactive and MN-inactive glycoprotein from human meconium by a conventional separation technique and designated its glycoprotein meconium-Vg antigen. More recently, &hide et al. (1993) have found that all of perchloric
INTRODUCTION
Recently, Ohkuma and Yanagi (1992) have elucidated that Vicia graminea lectin (VGA) and Vicia unijuga lectin (VUA) selectively recognize a small glyco (sialoglyco)-oligopeptide moiety (A) or (B) in glycoprotein molecules, respectively (Fig. I), and proposed that glycoproteins, which react with VGA and VUA, but do not react with anti-M and anti-N sera, are designated as a whole VGA- or VUA-binding (Vgu) glycoproteins, and Vgu glycoproteins, which also react with Arachis hypogaea lectin [PNA, anti-T (Thomsen-Friedenreich) lectin] and/or anti-T serum, are named Vgu glycoproteins with T activity. On the one hand, by then, a series of Vgu glycoproteins and Vgu glycoproteins with *Present address: Molecular Pharmacology Group, Tsukuba Research Laboratories, Nippon Glaxo Co., Ltd, 43 Wadai, Tsukuba, Ibaraki 300-43, Japan. tTo whom all correspondence should be addressed. (Received I8 January 1994; accepted 7 November 1994). 319
Noboru Uchide et al.
320
3
6
prepared from V. unijuga leaves by the method of Yanagi et al. (1990a). Influenza A and B viruses used in this work were PR8 strain and Osaka strain, respectively. Calibration reference substances were products of Sigma Chemical Co. (St Louis, MO., U.S.A.). VUA-Cellulofine was prepared by the method of Yanagi et al. (1990b). PNA-Agarose was obtained from Pharmacia Fine Chemical AB (Uppsala, Sweden). Asahipak GS-710 (vinyl alcohol polymer, different molecular weight range lo-lo4 kDa) column was a product of Asahi Chemical Industry Co., Ltd (Kawasaki, Kanagawa, Japan).
t
t
Chemical composition analysis
-Ser-Thr-Thr-
T:
Ts:
GalPl
----)
Gala1 4
-
Ser -Thr-
3GalNAca
+
1 -0
-
JGalNAca
---)
1 -0
-
NeuAcai NeuAcai Fig. 1. Small glyco (sialoglyco)-oligopeptide moiety (A) or (B) in glyoprotein molecules.
acid-soluble fractions (PASFs) from human meconiums reacted with VGA and VUA and very weakly reacted with both the T reagents, but did not react with anti-M and -N set-a.Therefore, from the studies of Springer et al. (1966) and Uchide et al. (1993) and the findings of Ohkuma and Yanagi (1992), it is possible to infer that human meconium contains Vgu glycoproteins and/or Vgu glycoproteins with T activity. To confirm the presence of Vgu glycoproteins and/or Vgu glycoproteins with T activity in human meconium, separation and characterization of these glycoproteins from human meconium by the systematic affinity chromatography, were studied in this work. MATERIALS
AND METHODS
Materials
PASF prepared from a mixture of meconiums of 22 newborn babies (blood-group type AMN, BMN, ABMN or OMN) by the perchloric acid method (Krupey et al., 1972) in the preceding study (Uchide et al., 1993), was used in this work. Anti-A, -B, -M and -N sera were purchased from Ortho Diagnostic System Inc. (Norcross, Ga, U.S.A.). Anti-H serum was prepared from Japanese eels (Angilla juponica) by the method of Yamamoto and Takizawa (I 98 1). Anti-T serum was prepared by immunizing rabbits with neuraminidase-treated human erythrocytes (OMN) according to the method of ijrntoft et al. (1985). VGA, PNA, Canavalia ensiformis lectin (Con A) and Phaseolus vulgaris lectin (PHA-E) were purchased from E-Y laboratories (San Mateo, Calif. U.S.A.). VUA was
Sialic acid, neutral sugar, amino sugars and amino acids were determined according to the methods used by Ohyama et al. (1993). HPLC
HPLC was carried out by using Rhodyne sample injector 7 125, Shimadzu constametric pumps LC-6A, Shimadzu photodiode array u.v.vis detector SPD-M6A and Asahipak GS-710 column (0.76 x 50 cm) under the conditions of a flow rate of 0.5 ml/min with 0.2 M phosphate buffer (pH 6.0) as an elution buffer. A 0.5 mg sample of V, VP or P fraction was dissolved in 50,ul of the same buffer and applied to the column. Eluates (each 0.25 ml) were collected and absorbance at 206 nm and reactivities for VUA and/or PNA of each of the eluates by ELISA were estimated. Apparent molecular weight (M,) estimation
M,s of Vl-V3, VPl, VP2 and Pm were estimated by HPLC. The relation between log molecular weight (Y, Da) of a sample (glycoprotein or reference substance) and its retention time (X, min) in HPLC was shown by the following equation: Y = -0.031071X2
+ 1.4127X -8.7525
This equation was calculated with Macintosh IIci computer (Apple Computer Inc., Cupertino, Calif., U.S.A.) using a soft program, CA-Cricket’“’ Graph IIITM (Computer Associates International Inc., U.S.A.). Ferritin (M, 450 kDa. Retention time (Rt) was 30.1 (min) in HPLC), apoferritin (M, 443 kDa, Rt 30.1) /3-amylase (M, 200 kDa, Rt 30.7) aldolase (M, 158 kDa, Rt 31.0) human IgG (M, 150 kDa, Rt 30.7) alcohol dehydrogenase (M, 150 kDa, Rt 31.4), ovalbumin (M, 45 kDa, Rt 31.8) and chymotrypsinogen (M, 25 kDa, Rt 32.4) were
Glycoproteins
used as calibration apparent molecular HPLC.
in human
reference substances in weight estimation by
Hemagglutination inhibitory assay Hemagglutination inhibitory assay of PASF and V, VP, P, Ul and U2 fractions was carried out by the method of Yanagi et al. (1990b). Enzyme-linked immunosorbent assay (ELBA) ELISA was carried out according to the method of Ueno et al. (1992). Systematic aJinity chromatography using VUAcellulojine column and PNA -agarose column of PASF In affinity chromatography, VUA-Cellulofine and PNA-Agarose columns previously equilibrated with buffer A (0.1 M Tris-HCl buffer (pH 7.2) containing 0.3 M NaCl) at 3-4°C were used and absorbance at 280 nm and reactivities for VUA and/or PNA of the effluent and eluate were estimated. Affinity chromatography and dialysis of the pooled effluents or eluates against distilled water were performed at 3-4°C. In affinity chromatography using VUA-Cellulofine column, the efflux and elution were carried out at in a flow rate of 3 ml/hr and the effluents or eluates (each 3 ml) were collected. A 30.0 mg sample of PASF was dissolved in 3.0 ml of buffer A and applied to a column (1 .O x 8.0 cm) of VUA-Cellulofine. The column was washed with buffer A and the effluents were pooled, dialyzed and lyophilized. The dry residue was
321
meconium
designated VUA-unbound fraction (Fig. 2). Bound matters on the column were eluted with 3.0 M KSCN in buffer A. The eluates were pooled, dialyzed and lyophilized. The dry residue was named VUA-bound fraction (Fig. 2). In affinity chromatography using PNA-Agarose column, the efflux and elution were all performed in a flow rate of 1 ml/hr and the effluents or eluates (each 1 ml) were collected. A 2.0 mg sample of VUA-bound fraction was dissolved in 1 ml of buffer A and applied to a column (1 .l x 2.0 cm) of PNA-Agarose. The column was washed with buffer A and the effluents were pooled, dialyzed and lyophilized. The dry residue was named VUA-bound and PNA-unbound (V) fraction (Fig. 3). Bound matters on the column were eluted with 0.2 M lactose in buffer A. The eluates were pooled, dialyzed and lyophilized. The dry residue was designated VUA-bound and PNA-bound (VP) fraction (Fig. 3). A 2.0 mg sample of VPunbound fraction was dissolved in 1 ml of buffer A and applied to a column (1.1 x 3.0 cm) of PNA-Agarose. The column was washed with buffer A and the effluents were pooled, dialyzed and lyophilized. The dry residue of two fractions were named VUA-unbound and PNA-unbound 1 (Ul) and 2 (U2) fractions, respectively (Fig. 4). Bound matters on the column were eluted with 0.2 M lactose in buffer A. The eluates were pooled, dialyzed and lyophilized. The dry residue was designated VUA-unbound and PNA-bound (P) fraction (Fig. 4).
VUA-bound fraction t I
0.0
0.0 10
0
20
30
40
Tube number Fig. 2. Affinity
chromatography
using
VUA-Cellulofine
column
of PASF
322
Noboru Uchide et al. VUA-bound and PNA-unboud l a2 (V) fraction
VUA-bound and PNA-bound (VP) fraction
I
I
0.9
nc lJ.0
0.3
0.0
0
10
20
: 0
30
Tube number Fig. 3. Affinity chromatography
using PNA-Agarose
RESULTS
Separation of the fractions of Vgu glycoprotein (V), Vgu glycoprotein with T activity (VP), T active glycoprotein (P) and other glycoproteins (171and U2) from PASF by a systematic afinity chromatography using VUA-Cellulojne column and PNA -Agarose column
PASF, which was prepared from a mixture of meconiums of 22 newborn babies (AMN, BMN, ABMN or OMN) was systematically subjected to affinity chromatography using
10
fraction.
VUA-Cellulofine column and PNA-Agarose column, as shown in Figs 2-4. In the first affinity chromatography using VUA-Cellulofine column, PASF into VUAwas separated unbound fraction and VUA-bound fraction (Fig. 2). In the second affinity chromatography using PNA-Agarose column, VUA-bound fraction was senarated into VUA-bound and PNAunbound &) fraction and VUA-bound and PNA-bound (VP) fraction (Fig. 3). In the third affinity chromatography using PNA-Agarose column, VUA-unbound fractions were separated
VUA-unbound and PNA-unbound 2 (U2) fraction
VUA-unbound and PNA-unbound 1 (Ul) fraction
0
column of VUA-bound
VUA-unbound and
20
L
,O
30
Tube number Fig. 4. Affinity chromatography
using PNA-Agarose
column of VUA-unbound
fraction.
Glycoproteins in human meconium
into VUA-unbound and PNA-unbound 1 (Ul) and 2 (U2) fractions and VUA-unbound and PNA-bound (P) fraction (Fig. 4). The fractions of V, VP, P, UI and U2 were obtained as a white solid, respectively. Yields of these fractions shown in Table 1 indicate that U2 fraction is the main fraction of PASF, P fraction is the second main fraction and each of the fractions of V and VP is the miner fraction. Chemical compositions and serological properties of the fractions of V, VP, P, UI and U2
All the fractions of V, VP, P, Ul and U2 were demonstrated to be glycoproteins containing sialic acid by their chemical composition analysis, as shown in Table 1. The notable features of the carbohydrate compositions of the five fractions were that these fractions contained a large amount of N-acetylgalactosamine, respectively, P fraction did not contain mannose and V, Ul and U2 fractions contained a very small amount of
323
mannose, respectively. The total contents of threonine and serine were considerably higher than that of aspartic acid in the five fractions. The reactivities of the five fractions with 13 hemagglutinins are presented in Table 2. All the fractions reacted with anti-A and -B sera, but did not react with anti-M and -N sera. Among these fractions, only Ul and U2 fractions reacted with anti-H serum. V fraction gave positive reactions with VGA and VUA and showed negative reactions with anti-T serum and PNA. VP fraction gave positive reactions with VGA, VUA and PNA and showed a very weak reaction with anti-T serum. V and VP fractions showed essentially the same reactivities with ConA, PHA-E and influenza viruses. P fraction gave a positive reaction with PNA, showed a very weak reaction with anti-T serum and gave negative reactions with VGA, VUA, ConA, PHA-E and influenza viruses. Ul and U2 fractions did not react with VGA and VUA and
Table 1. Yields and chemical compositions of V, VP, P, Ul and U2 fractions separated from PASF by a systematic affinity chromatography using VUA-Cellulofine column and PNA-Agarose column
Fraction
PASF”
VUA-bound and PNA-unbound (V)
Yield (mg/lOO mg of PASF)
151.9b
0.2
Carbohydrate composition (w/w,%) Sialic acid 9.3 Glucose n.d. Mannose 4.8 Galactose 22.8 Fucose 8.3 N-Acetylglucosamine 19.2 N-Acetylgalactosamine 9.1
2.8 1.2 0.8 8.4 2.8 27.8 19.7
Amino acid composition (mol/lOO mol) 10.4 7.5 Asp Thr 17.6 7.6 Ser 11.7 19.1 Glu 10.0 9.8 Pro 9.5 4.4 8.0 17.3 GUY .41a 7.8 8.9 1.1 n.d. CYS Val 6.1 4.6 Met 1.3 1.3 lie 3.4 2.5 Leu 7.6 4.2 1.7 1.7 Tyr Phe 3.7 2.3 4.6 4.1 LYS His 2.8 2.8 2.4 1.8 Arg nd., not detected. “cited from the paper of Uchide et al. (1993). bmg/g of wet human meconium.
VUA-bound and PNA-bound (VP)
VUA-unbound and PNA-bound (P)
0.2
19.0
VUA-unbound VUA-unbound and and PNA-unbound I PNA-unbound 2 (LJ2) u-J11 13.2
7.0
3.3
8.1
nd.
nd. n.d.
nd.
4.6 17.2 7.1 11.8 12.3 4.2 24.8 16.0 4.4 10.8
28.2 4.4 22.9 16.4
1.6 21.3 8.9 18.7 11.9
5.5
9.7
4.5 25.4 17.7 4.8 11.6 6.5 8.3
nd.
nd.
4.5 0.2 2.7 4.2
4.8
5.5
I.0
1.9 3.3
10.1
1.1 1.6 1.8 1.7 2.3
2.4 3.6 0.8 1.3 2.2 2.5 2.7
24.4 11.9 7.6 10.3 8.3 6.1 0.7
5.1 1.5 2.3 2.3 1.6 1.8
54.0
7.0 0.2 1.4
5.9 2.8 18.2
16.4 IO." 11.1 13.0 Il.’ 1.7 I I.2 ?i 0.4 4.4
I.3 27 6.3
17 31 41
12 2s
Noboru Uchide et al.
324
Table 2. Hemagglutination inhibition activities of V, VP, P, UI and U2 fractions separated from PASF by a systematic affinity chromatography using VUA-Cellulofine column and PNA-Agarose column
Fraction
PASF”
Hemagglutinin
(titer = 8)
Anti-A serum Anti-B serum Anti-H eel serum Anti-M serum Anti-N serum Anti-T serum Arachis hypogaea (PNA, anti-T lectin) Vi& gruminea (VGA) Vicia unijugu (VUA) Canavalia
1 2 1250
n.i. n.i. v.w.r. v.w.r.
VUA-bound VUA-bound VUA-unbound VUA-unbound VUA-unbound and and and and and PNA-unbound PNA-bound PNA-bound PNA-unbound 1 PNA-unbound 2 (V (VP) m W1) (W Minimum concentration @g/ml) of fraction giving complete inhibition 156
10
78
20
n.i. n.i. n.i. n.i. n.i.
n.i. n.i. n.i. V.W.T.
n.i. n.i. n.i. v.w.r.
39
313 78
20
39 39
1 10
5 5 313
1250
156
xi. n.i. v.w.r. v.w.r.
n.i. n.i. v.w.r. v.w.r.
n.i. n.i. n.i.
n.i. n.i. 1%
n.i. xi. 1250
ensiformis
5000 1250 1250
n.i.
10 n.i.
vulgaris
1250
2500
1250
n.i.
625
n.i.
313 625
625 2500
1250 1250
n.i. n.i.
39 39
1250 1250
(Con A) Phaseolus
(PHA-E) Influenza A virus Influenza B virus
n.i., no inhibition of hemagglutination at 5000 pg/ml. v.w.r., a very weak reaction. “cited from the paper of Uchide et al. (1993)
gave very weak reactions with both the anti-T reagents, while reacted with ConA and Influenza viruses. HPLC analyses of the fractions of V, VP and P In HPLC, V fraction gave numerous VUA-reactive peaks including the three main peaks, VI, V2 and V3, as shown in Fig. 5. The elution positions of VUA-reactive peaks did not agree with those of the peaks of absorbance at 206 nm. Apparent M,s of
19,364 kDa, 5491 kDa and 1871 kDa were found for VI, V2 and V3 by HPLC, respectively. VP fraction gave VP1 and VP2 peaks in HPLC, as shown in Fig. 6. The elution positions of VUA-reactive peaks agreed substantially with those of PNA-reactive peaks, but differed from those of the peaks of absorbance at 206 nm. Apparent M,s of 462 kDa and 152 kDa were found for VP1 and VP2 by HPLC, respectively. As seen in Fig. 7, P fraction gave the main PNA-reactive peak, Pm, with M, of
Tube number Fig. 5. HPLC analysis of V fraction
Glycoproteins in human meconium
325
Tube number Fig. 6. HPLC analysis of VP fraction
11,680 kDa, in HPLC. The elution position of Pm differed from those of peaks of absorbance at 206nm. DISCUSSION
In the preceding paper (Uchide et al. 1993), we have reported that PASF extracted from a mixture of meconiums of 22 newborn babies with blood group AMN, BMN, ABMN or OMN type by the perchloric acid method (Krupey et al., 1972), reacted with VGA and VUA and also reacted very weakly with PNA and anti-T serum, but did not react with anti-M
and -N sera. In this work, to clarify the presence of Vgu glycoproteins and/or Vgu glycoproteins with T activity in human meconium, PASF was subjected to a systematic affinity chromatography using VUA and PNA as immobilized ligands. First, PASF was separated into VUAunbound fraction and VUA-bound fraction in affinity chromatography with VUA-Cellulofine column (Fig. 2). Secondly, VUA-bound fraction was separated into VUA-bound and PNAunbound (V) fraction and VUA-bound and PNA-bound (VP) fraction in affinity chromatography with PNA-Agarose column (Fig. 3). Thirdly, VUA-unbound fraction was separated
Pm
Tube number Fig. 7. HPLC analysis of P fraction
326
Noboru Uchide et al.
into VUA-unbound and PNA-unbound I (Ul) VUA-reactive and MN-inactive glycoproteins and 2 (U2) fractions and VUA-unbound and have been designated Vgu glycoproteins and PNA-bound (P) fraction in affinity chromaVgu glycoproteins, which also react with PNA tography with PNA-Agarose column (Fig. 4). and/or anti-T serum, have been named Vgu The five fractions, V, VP, P, Ul and U2, glycoproteins with T activity (Ohkuma and were sugar rich glycoprotein fractions with Yanagi, 1992). Accordingly, it is apparent that V and VP fractions are a Vgu glycoprotein 60.0, 63.5, 75.2, 70.5 and 52.4% carbohydrate, respectively. V fraction contained N-acetylfraction and a fraction of Vgu glycoprotein with galactosamine and serine in high percentages, T activity, respectively, and P fraction is a mannose in a very low percentage and aspartic T-active glycoprotein fraction, while Ul and U2 acid in a low percentage (Table 1). The fractions are a glycoprotein fraction without N-acetylgalactosamine content was much having Vgu and T activities, respectively, and higher than the mannose content in V and VP that human meconium contains Vgu glycofractions which contained aspartic acid in a low proteins, Vgu glycoproteins with T activity and percentage, respectively. P fraction contained T-active glycoproteins. N-acetylgalactosamine and serine in high perTo confirm the glycoprotein composition of centages and threonine in a very high percent- V, VP, and P fractions, these fractions were age, but did not contain mannose (Table 1). analyzed by HPLC (Figs 5-7). The analysis It is well known that the N-acetylgalactosamine results indicate that V fractions contained three residue is a characteristic component of O-gly- Vgu glycoproteins, Vl glycoprotein with M, of cosidically linked saccharide chain (mucin-type about 19,400 kDa, V2 glycoprotein with M, sugar chain) and the serine and threonine of 5500 kDa and V3 Glycoprotein with M, of residues are essential for the formation of O-gly1900 kDa, VP fraction consisted of two kinds cosidic linkage, on the one hand, the mannose of Vgu glycoprotein with T activity, VP1 glycoresidue is characteristically contained in the protein with M, of about 460 kDa and VP2 N-glycosidically linked saccharide chain (as- glycoprotein with M, of 150 kDa and P fraction paragine-linked sugar chain) and the asparagine composed of several T-active glycoproteins (aspartic acid) residue is the essential amino acid including the main glycoprotein (Pm glycoprotein) with M, of about 11,700 kDa. VI-V3, component of N-glycosidic linkage (Kobata, VPl, VP2 and Pm glycoproteins seem to be 1984). Consequently, the carbohydrate composition analyses of the fractions of V, VP and P a kind of mucin-type glycoproteins (Feizi et al., seem to show that the mucin-type sugar chains 1984), because these glycoproteins bear high are more remarkably abundant than the as- molecular weight and abundant mucin-type paragine-linked sugar chains in V and VP frac- sugar chains, respectively, as described above. tions and, on the other hand, P fraction Proteins or antigens, which occur in common abundantly contains only the mucin-type sugar both in carcinoma tissues and fetus tissues, were chains. termed carcinofetal proteins (Nishi, 198 l), All of PASF and its five fractions, V, VP, P, oncofetal proteins (Yachi, 1981) or oncofetal Ul and U2, showed no reactions with anti-M antigens (Waldman and Herberman, 1982). On and -N sera. V and VP fractions reacted with the one hand, before then, meconium-Vg glycoVGA and VUA, and PASF reacted weakly with protein had been separated from human the same two lectins, but P, Ul and U2 fractions meconium (Springer et al., 1966) and then did not react with the same two lectins. VP and Vl-V3 glycoproteins (Vgu glycoproteins), VP1 P fractions reacted with PNA, and PASF and and VP2 glycoproteins (Vgu glycoproteins with the fractions of U 1 and U2 reacted very weakly T activity) and Pm glycoprotein (T-active glycowith PNA, but V fraction did not react with the protein) were isolated from human meconium same lectin. V fraction did not react with anti-T in this work. On the other hand, it has been serum, and PASF and the fractions of VP, P, U 1 found that all 21 kinds of PASFs from human and U2 reacted very weakly with the same intact tissues (the brain, submaxillary gland, anti-serum (Table 2). Therefore, the serological esophagus, stomach, small intestine, large study described here seems to show that Vgu intestine, liver, gallbladder, pancreas, lung, activity of PASF was concentrated in its V and heart, spleen, thymus gland, kidney, bladder, VP fractions and T activity of PASF was adrenal gland, prostatic gland, ovary, skeletal concentrated in its VP and P fractions. As muscle and skin) and human normal serum described in introduction, recently, VGA- or gave no reactions with VGA, VUA, PNA and
Glycoproteins in human meconium
anti-M and -N sera (Ohyama et al., 1989) and reported that a series of Vgu glycoproteins, Vgu glycoproteins with T activity and T-active glycoproteins were isolated from human liver metastases of pancreas carcinoma (Ueno et a[., 1992) and breast carcinoma (Ishigure et al., 1991), cyst fluids of human ovarian clear cell carcinoma (Yanagi et al., 1990b) and ascitic fluids of primary ovarian cancer patients (Hayashi et al., 1990) and also detected in human primary hepatocellular carcinoma (Otsuka et al., 1992) human liver metastases of sigmoid colon carcinoma (Otsuka et al., 1988, 1991a) and ascitic fluids of liver cancer patients (Otsuka et al., 1991b). Therefore, it may be concluded that Vgu glycoproteins, Vgu glycoproteins with T activity, T-active glycoproteins and meconium-Vg glycoprotein are new carcinofetal glycoproteins, oncofetal glycoproteins or oncofetal antigens, respectively. REFERENCES
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