| Glycobiology | Pages |
Studies on the binding site of the galactose-specific agglutinin PA-IL from Pseudomonas aeruginosa
Introduction
Results
Discussion
Materials and methods
Acknowledgments
Abbreviations
References
Studies on the binding site of the galactose-specific agglutinin PA-IL from Pseudomonas aeruginosa
The binding properties of Pseudomonas aeruginosa agglutinin-I (PA-IL) with glycoproteins (gps) and polysaccharides were studied by both the biotin/avidin-mediated microtiter plate lectin-binding assay and the inhibition of agglutinin-glycan interaction with sugar ligands. Among 36 glycans tested for binding, PA-IL reacted best with two glycoproteins containing Gal[alpha]1->4Gal determinants and a human blood group ABO precursor equivalent gp, but this lectin reacted weakly or not at all with A and H active gps or sialylated gps. Among the mammalian disaccharides tested by the inhibition assay, the human blood group Pk active Gal[alpha]1->4Gal, was the best. It was 7.4-fold less active than melibiose (Gal[alpha]1->6Glc). PA-IL has a preference for the [alpha]-anomer in decreasing order as follows: Gal[alpha]1->6 > Gal[alpha]1->4 > Gal[alpha]1->3. Of the monosaccharides studied, the phenyl[beta] derivatives of Gal were much better inhibitors than the methyl[beta] derivative, while only an insignificant difference was found between the Gal[alpha] anomer of methyl- and p-NO2-phenyl derivatives. From these results, it can be concluded that the combining size of the agglutinin is as large as a disaccharide of the [alpha]-anomer of Gal at nonreducing end and most complementary to Gal[alpha]1->6Glc. As for the combining site of PA-IL toward the [beta]-anomer, the size is assumed to be less than that of Gal; carbon-6 in the pyranose form is essential, and hydrophobic interaction is important for binding.
Key words: binding properties/combining sites/glycoproteins/ lectin/Pseudomonas aeruginosa
Introduction
Pseudomonas aeruginosa (PA) is an opportunistic pathogenic bacterium attacking immunocompromised patients. Its mainly intracellular PA-I and PA-II lectins, and other adhesive molecules, including sialic acid-binding, ganglioside-binding, and hydrophobic adhesins, play an important role in causing human infection and serious tissue damage (Gilboa-Garber, 1972, 1982; Ramphal et al., 1984; Garber et al., 1985; Gilboa-Garber and Garber, 1992). PA-IL, which is a tetrameric protein with an average subunit molecular mass of approximately 13,000 Da, and tends to aggregate in the purified state, is a d-Gal specific lectin (Gilboa-Garber, 1972; Gilboa-Garber et al., 1977). This agglutinin was found to have a stronger affinity for Gal[alpha]1-> than GalNAc (Gilboa-Garber, 1972) and a great affinity for B, Pk, and P1 blood group types of papain-treated erythrocytes (Gilboa-Garber et al., 1994). It has also been reported that the lectin binds strongly to terminal and nonsubstituted Gal[alpha]1->3Gal or Gal[alpha]1->4Gal structures of glycosphingolipids and neoglycoproteins (Lanne et al., 1994). However, the methods used to characterize its affinity were mainly by hemagglutination inhibition and equilibrium dialysis (Gilboa-Garber et al., 1994) and TLC plate assay (Lanne et al., 1994); the number and type of glycoforms used was limited, and the information of the relative potency of sugar inhibitors was incomplete. Thus, more detailed information about the fine specificity and binding properties of PA-IL may ultimately help us to understand the spreading or adhesive mechanisms of P.aeruginosa in humans. Therefore, we have examined (1) the combining site of PA-IL by our recently established, biotin/avidin mediated microtiter plate lectin-enzyme binding assay (Duk et al., 1994); (2) its interactions with many well known glycoproteins and polysaccharides; and (3) its relative affinity for carbohydrates using a series of sugar inhibitors.
The results indicate that this lectin reacts strongly with Gal[alpha]1->4Gal containing glycoproteins, many blood group ABH precursor glycoproteins and/or their equivalents. The combining size of the agglutinin is as large as that of a disaccharide having the [alpha]-anomeric configuration of Gal at the nonreducing end and is most complementary to melibiose(Gal[alpha]1->6Glc). The combining size of PA-IL toward the [beta]-anomer is less than that of Gal and hydrophobic interaction is important for the binding.
Results
Lectin-glycoform interaction
The avidity of Pseudomonas aeruginosa agglutinin-I lectin (PA-IL) for glycoproteins (gps) and Pneumococcus type XIV polysaccharide, studied by a microtiter plate lectin-enzyme binding assay, is summarized in Table I and selected interaction profiles are shown in Figure 1. Among 36 glycans tested for binding, PA-IL reacted best with two Gal[alpha]1->4Gal containing glycoproteins: a human blood group P1 active glycoprotein purified from sheep hydatid fluid (Figure 1a, Table I), and the asialo bird nest glycoprotein (structure I; Figure 1b and Table I); it also bound strongly to a human blood group ABO precursor equivalent glycoprotein (Beach phenol insoluble P-1) (Structure II, Figure 1a, Table I). Only 0.1 µg of glycoprotein was required to interact with 0.25 µg of lectin to reach an A405 above 2.5 after 2 h. The percentage of the adsorbance of the glycoprotein on the microtiter plate has not been established. Thus, the glycoproteins required to reach maximum interaction should be equal to or less than 0.1 µg. The lectin also reacted well with many other blood group ABO precursor glycoproteins (cyst OG 10% 2× PPT) (Figure 1b), or equivalents (Table I) (1st Smith of 10% 2× cyst MSS, Mcdon P-1 and Tighe O P-1) (Figure 1c), and B active gps (cyst Beach phenol insoluble, Horse 4 and cyst 19) (Figure 1a,c).
Figure The blood group A, H, or Leb substances and sialic acid containing glycoproteins from mammalian salivary glands tested were either weakly active or inactive (Table I). These included bird nest glycoprotein (Figure 1b), cyst MSS 10% 2×(A1), cyst Mcdon (A1), cyst JS phenol insoluble(H), cyst Tighe phenol insoluble (H+Leb), hog gastric mucin #4(A+H) (Table I), HOC 350, RSL, BSM, PSM, and ASG. Asialo glycoproteins from salivary glands which contain a large number of exposed T, Tn, and/or II determinants were either inactive or weakly active (ranging from - to two ++, Table I). Neither fetuin, human [alpha]1-acid glycoprotein nor their asialo products (Table I) reacted with PA-IL. Table I. Removal of sialic acid from the PA-IL nonreactive bird nest glycoprotein led to a substantially increased activity (Figure 1b); after Smith degradation or mild acid hydrolysis at pH 1.5, 100°C for 2 h (the nondialyzable fraction was defined as P-1 fraction), the poorly reactive blood group A and H substances increased in reactivity (Figure 1c). Similarly, the binding of Beach phenol insoluble, a blood group B active glycoprotein (Figure 1a), increased from +++ to +++++ after mild acid hydrolysis (Table I). Table II
Table III.
Curve in Figure 1
Glycoprotein or polysaccharide
Lectin determinantsb (blood group specificity)
O.D.405
Avidityc
a
Sheep hydatid cyst gp
E(P1)
3.0
+++++
a
Cyst Beach phenol insoluble
B
1.85
+++
a
Beach P-1
T,Tn,I,II
2.87
+++++
b
Bird nest gp (BN)
II,E,T,F
0.14
-
b
Asialo BN
II,E,T,F
2.64
+++++
b
Cyst OG 10% 2x PPT
B,I/II(Ii)
2.29
++++
c
Cyst 19(B+)
B
1.53
+++
Cyst Tijani 20% of 2nd 10%
B,I/II(Ii)
0.82
++
c
Horse 4(B+)
B
1.41
++
c
Cyst MSS 10% 2×
Ah(A1)
0.05
-
c
1st Smith MSS
T,Tn,I,II
1.44
++
c
Cyst Mcdon
Ah
0.02
-
c
Mcdon P-1
T,Tn,I,II
1.71
+++
Cyst JS phenol insoluble
H
0
-
c
Cyst Tighe phenol insoluble
H,Leb
0
-
c
Tighe O P-1
I/II(Ii)
1.99
+++
Cyst 9 phenol insoluble
Ah(A1)
0
-
Cyst 14 phenol insoluble
Ah(A2)
0.01
-
Cyst MSM 10% PPT
Ah
0.06
-
MSS 10%
Ah(A1)
0.05
-
Hog gastric mucin #4
Ah,H
0
-
Armadillo submandibular gp (ASG)
Sialy Tn,Tn
0.15
-
Asialo ASG
Tn
0.02
-
d
Cyst HOC 350
T,Tn,I,II
0.02
-
d
Asialo HOC 350
T,Tn,I,II
0.91
++
d
Porcine salivary gp(PSM)
A,Ah,T,Tn
0.01
-
d
Asialo PSM
A,Ah,T,Tn
0.63
+
d
Bovine salivary gp-major (BSM)
T,Tn
0.04
-
d
Asialo BSM
T,Tn
0.84
++
d
Rat sublingual major-gp (RSL)
II
0.02
-
d
Asialo RSL
II
0.45
+
Pneumococcus type 14 PS
II
0.11
-
Human [alpha]1-acid gp
II
0.16
-
Asialo human [alpha]1-acid gp
II
0.18
-
Fetuin
II,T
0.01
-
Asialo fetuin
II,T
0.11
-
Curve in Fig. 1
Blood group active glyco-protein purified from human
Human blood group determinant present
Sugar added to Structure II
Site of addition to Structure II
b
Cyst OG 10% 2×PPT
Cyst Tij 20% of 2×10%
c
MSS 1st Smith2
Ii
a
Beach phenol insoluble
Pneumococcus type XIV
None
a
P-12
polysaccharide
c
Mcdon P-12
antigenic determinant
c
Tighe O P-12
d
Asialo HOC 3502,3
H, Leb
LFuc[alpha]1->2
(5),(7),(9),
c
Tighe phenol insoluble
LFuc[alpha]1->4
(6),(8),(10),
LFuc[alpha]1->3
(11) and (12)
c
MSS, native
GalNAc[alpha]1->3
(1),(2),(3)
A1 or A2
and as in
and/or (4)
Cyst 9
H, and Leb
(5), (7), (9)
Cyst 14
and (12)
c
Cyst Mcdon
Gal[alpha]1->3 and
(1),(2),(3)
a
Beach phenol insoluble
B
as in H and
and/or (4),(5),
Leb
(7),(9),(6),(8)
and (12)
Order of activity
Curve in Fig. 2
Inhibitor
Quantity giving 50% inhibition (nmol)
Reciprocal of relative potencyb
1
A
Phenyl[beta]Gal
0.7
57.1
2
A
p-NO2-Phenyl[beta]Gal
2.0
20.0
3
B
Melibiose(Gal[alpha]1->6Glc)
3.0
13.3
4
B
Stachyose
(Gal[alpha]1->6Gal[alpha]1->6Glc[beta]1-> 2DFruf) 7.0
5.7
5
A
Gal[alpha]1->3Gal[alpha]1->methyl
8.5
4.7
6
B
Raffinose
(Gal[alpha]1->6Glc[beta]1->2DFruf) 9.0
4.4
7
A
p-NO2-Phenyl[alpha]Gal
14.0
2.9
8
A
Methyl[alpha]Gal
15.0
2.7
9
A
Methyl[beta]Gal
18.0
2.2
10
A
Gal[alpha]1->4Gal (E)
22.0
1.8
11
A, B
Gal
40.0
1.0
12
A
Gal[alpha]1->3Gal (B)
50.0
0.8
13
B
GalNAc
80.0
0.5
14
A, B
Gal[beta]1->4Glc (L)
80.0
0.5
15
B
Gal[beta]1->3Ara
350
0.1
16
B
d-Fuc
1800
0.02
| Inhibitor | Maximum amount of inhibitor used (nmol) | Percentage inhibition (%) |
| Gal[beta]1->3GlcNAc (I) | 66.8 | 40.0 |
| Gal[beta]1->4GlcNAc (II) | 65.2 | 25.0 |
| Gal[beta]1->3GalNAc (T) | 65.0 | 16.7 |
| Gal[beta]1->3GalNAc[alpha]1-> benzyl(Ta) | 52.5 | 2.5 |
| p-NO2-Phenyl[alpha]GalNAc | 61.0 | 9.9 |
| p-NO2-Phenyl[beta]GalNAc | 63.3 | 9.9 |
| Glc | 7000.0 | 9.0 |
| Man | 9333.3 | 8.7 |
| Maltose | 4870.0 | 7.4 |
| GlcNAc | 4783.3 | 0 |
| Sucrose | 4870.0 | 0 |
| l-Fuc | 7740.00 | 0 |
| l-Ara | 1597.5 | 0 |
As shown in Table I, this lectin exhibited variations in avidity for several blood groups: B, I, and/or precursor equivalent substances from ++ to ++++ (Figure 1b,c). OG 10% 2× PPT reacted very well (++++) with the lectin, but less so (++) with Horse 4(B+) and cyst Tijani 20% of 2nd 10%. These results are most likely due to the different degrees of substitutions and their configuration that affect lectin-carbohydrate binding.
Inhibition of lectin-glycan interaction
The ability of various sugars to inhibit the binding of PA-IL by the human blood group P1-active glycoprotein purified from sheep hydatid cyst fluid is shown in Figure 2, and the amounts of different ligand required for 50% inhibition of the lectin-glycan interaction are listed in Table III.
Figure Among the oligosaccharides tested for inhibition of interaction, melibiose (Figure 2, curve 3, Gal[alpha]1->6Glc) was the best; it was 13.3-, 3.0-, and 2.3-fold more active than Gal (curve 11), raffinose (curve 6, Gal[alpha]1->6Glc[beta]1->2DFruf), and stachyose (curve 4, Gal[alpha]1->6Gal[alpha]1-> 6Glc[beta]1->2DFruf), respectively. The human blood group Pk active disaccharide Gal[alpha]1->4Gal (curve 10) was 1.8 times more active than Gal, but 7.3 times less active than melibiose; human blood group B active disaccharide (curve 12, Gal[alpha]1->3Gal) and lactose (curve 14, Gal[beta]1->4Glc) were about 0.8 and 0.5 that of Gal, respectively; 67 nmol of Gal[beta]1->3GlcNAc(I) and 65.2 nmol of Gal[beta]1->4GlcNAc(II) produced about 40% and 25% of inhibition, respectively (Table IV). Of the monosaccharides studied, phenyl[beta]Gal (Figure 2, curve 1) was the best inhibitor, which was 57, 2.9, and 4.3 times more active than Gal, p-NO2-phenyl[beta]Gal and melibiose, respectively. As shown in Table III, the phenyl[beta] derivative of Gal was a better inhibitor than the methyl[beta] derivative (Figure 2, curve 9), while insignificant difference was observed between the [alpha] anomer of methyl- and p-NO2-phenylGal (curves 7 and 8). Gal was about twice as potent inhibitor compared to GalNAc (curve 13), indicating that the N-acetamido group at C-2 in the pyranose ring interferes with interaction. DFuc (curve 16) showed 1/50 of Gal activity (curve 11), suggesting that the OH group on C-6 participates in binding; l-Ara, which has the same configuration as dGal but lacks the CH2OH of C6, was inactive up to a 40-fold higher amount than the molar amount of Gal used, indicating that the CH2OH of C6 is necessary for binding. p-Nitrophenyl[alpha]GalNAc and p-nitrophenyl[beta]GalNAc were inactive up to 63.3 nmol. GlcNAc, Man, Glc, lFuc, maltose, and sucrose were also tested from 4870 up to 7740 nmol, but only negligible or no inhibition was observed (Table IV).
Discussion
In this study, we examined the binding activity of PA-IL using a recently described method in which the lectin was biotinylated and binding was detected with alkaline phosphatase-conjugated avidin (Duk et al., 1994; Lisowska et al., 1996; Wu et al., 1997b). A wide range of partially characterized glycoprotein preparations were examined for binding and this is related to the presence of certain oligosaccharide structures defined in other studies. This study is also examining the inhibition of lectin binding with a variety of defined oligosaccharides in solution. Overall, the results are consistent with previous studies (Garber et al., 1992; Lanne et al., 1994).
During the past two decades, quantitative precipitin (QPA) and precipitin-inhibition (QPIA) studies of lectins with glycoforms have been successfully used to investigate the binding properties of lectins (Wu et al., 1980, 1988, 1992, 1993, 1994; Wu and Sugii, 1988, 1991). Such approaches can provide insight into the specificities and size parameters of the combining sites of lectins. However, these two assays have some limitations. They can not be used to study all of the lectin combining sites, as relatively high amounts of reagents (lectin, glycoproteins, and inhibitors) are required. To characterize the binding properties of lectins, such as described in our previous publications using both QPA and QPIA (Wu et al., 1988, 1992, 1995), over 10 mg of lectin has to be used. To solve this problem, we developed a new and highly sensitive microtiter plate lectin-enzyme binding assay (Duk et al., 1994). The lectin amount required for this assay is about 1/10 to 1/1000 of that (amount) required for the QPA and QPIA methods.
PA-IL is a galactophilic lectin (Gilboa-Garber, 1972; Gilboa-Garber et al., 1991, 1994; Garber et al., 1992) which exhibits preferential avidity for certain P related antigens. In this paper, we expand the scope of the study of its binding property and examine: (1) the combining site of this agglutinin by the lectin-enzyme binding microtiter plate system (Duk et al., 1994); and (2) its interactions with dozens of well known glycoproteins and polysaccharides (Table I; Structures I, II). From the present results, it can be concluded that the microtiter plate method chosen here is very useful to characterize the binding property of PA-IL.
Among 36 glycans tested for binding, two Gal[alpha]1->4Gal containing glycoproteins (Structure I) and human blood group ABO precursor glycoproteins or equivalents (Structure II; Figure 1a,c) reacted well with PA-IL. These results imply that Gal[alpha]1->4Gal and Gal[beta]1-> linked at the nonreducing end play important roles in binding. PA-IL also reacted strongly with many other B active gps, but weakly or not at all with A and H active glycoproteins or sialylated gps (Figure 1c,d). This can be explained by the masking effect of LFuc[alpha]1->, GalNAc[alpha]1-> and sialic acid, on the nonreducing terminal Gal[beta]1->.
Mild acid hydrolysis (pH 1.5, 100°C for 2 h) removes most of the LFuc[alpha]1-> linked at end groups and some blood group A and B active oligosaccharide side chains (Kabat et al., 1948; Beiser and Kabat, 1952; Leskowitz and Kabat, 1954; Allen and Kabat, 1959), and Smith degradation removes almost all nonreducing terminal sugars (Table I; Wu et al., 1982). Consequently, the reactivity of these treated products, or of the asialo glycoproteins, with this agglutinin should significantly increase (Table II). The Gal[beta]1-> determinant in these glycoproteins is similar to Bombay type erythrocytes(Oh) that show good reactivity with the lectin (Gilboa-Garber et al., 1991, 1994). Gilboa-Garber (1972) found that PA-IL strongly agglutinates papain-treated erythrocytes, which are devoid of the T or MN-bearing glycoproteins, indicating that this lectin (unlike PNA) does not exhibit exclusive specificity to the T determinant.
Among the mammalian disaccharides tested for inhibition of lectin-glycoform interaction, the human blood group Pk active disaccharide Gal[alpha]1->4Gal was most reactive; it was 1.8 times more active than Gal, but 7.4, 2.4, and 3.2 times less active than melibiose, raffinose, and stachyose, respectively, indicating that the combining size of this lectin is smaller than that of trisaccharides and most complementary to melibiose (Gal[alpha]1->6Glc). The inhibitory power of the human blood group B active disaccharide (Gal[alpha]1->3Gal) and lactose (Gal[beta]1->4Glc) was about 0.8 and 0.5 times that of Gal, respectively, indicating that the inhibitory affinity of Gal can be interfered with by subterminal [beta]1->4Glc or [alpha]1->3Gal. Furthermore, Gal[beta]1->3GlcNAc and Gal[beta]1->4GlcNAc were poor inhibitors (Table IV). Thus, the reactivity in decreasing order of the [alpha]-anomeric form of Gal can be summarized as follows: Gal[alpha]1->6 > Gal[alpha]1->4 > Gal[alpha]1->3. Subterminal sugars which are linked to the [beta]-anomer of Gal reduce the reactivity indicating that the size of the combining site of PA-IL toward the [beta]-anomer is less than Gal.
The variations in PA-IL binding to Gal and its derivatives are shown in Table III. It can clearly be seen that phenyl-[beta] derivatives of Gal are much better than its methyl-[beta] derivatives in binding PA-IL, while an insignificant difference was found between the Gal[alpha] anomer of the methyl- and p-NO2-phenyl derivatives. All these features illustrate the unique binding property of this agglutinin. By combining the present results with previously reported data (Gilboa-Garber, 1972; Gilboa-Garber et al., 1977, 1991, 1994; Garber et al., 1992), it is concluded that the combining site of the PA-I agglutinin can accommodate glycans as large as a disaccharide of the [alpha]-anomer of Gal at the nonreducing end and is most complementary to melibiose (Gal[alpha]1->6Glc). For the combining site of PA-IL toward the [beta]-anomer, the size is less than that of Gal, in which the epimer of carbon-4 and the presence carbon-6 are essential and hydrophobic interaction is important for the binding.
Materials and methods
The lectin
The Pseudomonas aeruginosa (PA-I) lectin was purified from extracts of the bacterium (ATCC 33347) as previously described (Gilboa-Garber, 1982). Heat-labile proteins were removed by heating at 70°C for 15 min and centrifugation. The lectin was precipitated from the supernatant by 60% saturation of ammonium sulfate at 4°C and was further purified by Sepharose 4B column affinity chromatography and elution with 0.2 M Gal.
Biotinylation of the lectin
For PA-IL biotinylation by biotinamidocaproate-N-hydroxy-succinimide ester (biotin ester, purchased from Sigma Chemical Co., St. Louis, MO), the lectin (200 µg/250 µl PBS) was mixed with 400 µl of the biotin ester solution (100 µg biotin ester per 200 µg lectin) and left for 30 min at room temperature. The biotinylated lectin was dialyzed for 2-3 h against ddH2O and overnight against TBS. After dialysis, the sample volume was adjusted to 1 ml with TBS and 20 µl of 5% sodium azide was added (equivalent to 200 µg/ml lectin at final concentrations in 0.1% NaN3; Duk et al., 1994).
Glycoproteins and polysaccharide
The human blood group P1-active substance, purified from sheep hydatid cyst glycoprotein, and HOC 350, a sialic acid rich glycoprotein, isolated from human ovarian cyst fluid (Pusztai and Morgan, 1961), were kindly provided by Dr. W. M. Watkins, University of London, Royal Postgraduate Medical School, Hammersmith Hospital, London. The sheep hydatid cyst P1-active glycoprotein was isolated from sheep hydatid cyst by extraction with 95% w/v phenol. The fraction was phenol insoluble and precipitated between 37-40% EtOH (Cory et al., 1974; Morgan and Watkins, 1964). HOC 350 was prepared by the method described by Pusztai and Morgan (1961) and Morgan (1965). The mucus glycoprotein (native BN), the so-called nest-cementing substance, from the salivary gland of Chinese swiftlets (genus Collocalia), was extracted with ddH2O at 60°C for 20 min from the commercial bird nest (BN) (Wieruszeski et al., 1987). The other purified blood group substances used were prepared from human ovarian cyst fluid, and from horse stomach (Beiser and Kabat, 1952; Kabat, 1956; Lloyd and Kabat, 1968; Vicari and Kabat, 1969, 1970; Maisonrouge-McAuliffe and Kabat, 1976; Newman and Kabat, 1976). The blood group substances were purified from human ovarian cyst fluid by digestion with pepsin and precipitation with increasing concentration of ethanol; the dried ethanol precipitates were extracted with 90% phenol, the insoluble fraction being given after the name of the blood group substance (e.g., Cyst Beach phenol insoluble). The supernatant was fractionally precipitated by addition of 50% ethanol in 90% phenol to the indicated concentrations. The designation 10 or 20% (ppt) denotes a fraction precipitated from phenol at an ethanol concentration of 10 or 20%; 2× signifies that a second phenol extraction and ethanol precipitation were carried out (e.g., Cyst OG 20% 2×). Regardless of their A, B, H, or Leb activity, the purified water-soluble blood group substances have a similar overall structure. They are polydispersed macromolecules (Mr 200,000-1,000,000) of similar composition (75 to 85% of carbohydrate, 15-20% protein). They consist of multiple heterosaccharide branches attached by glycosidic linkages at their internal reducing ends to serine or threonine of the polypeptide backbone (Beiser and Kabat, 1952; Kabat, 1956; Allen and Kabat, 1959; Lloyd and Kabat, 1968; Vicari and Kabat, 1969, 1970; Maisonrouge-McAuliffe and Kabat, 1976; Wu et al., 1982, 1984).
In general, the P-1 fractions represent the nondialyzable portion of the blood group substances after mild hydrolysis at pH 1.5-2.0 for 2 h which removed most of the l-fucopyranosyl end groups, as well as some blood group A and B active oligosaccharide side-chains (Kabat et al., 1948; Beiser and Kabat, 1952; Leskowitz and Kabat, 1954; Allen and Kabat, 1959). The first Smith-degraded products of blood group A active substances (MSS 10% 2×, Structure II), in which almost all of the sugar groups at the nonreducing ends were removed (Wu et al., 1984), were prepared as described previously (Lloyd and Kabat, 1968; Vicari et al., 1970; Wu et al., 1982).
The Pneumococcus type XIV polysaccharide was prepared as described previously (Howe et al., 1958; Lindberg et al., 1977). Fetuin (Gibco, Grand Island, New York), which is the major glycoprotein in fetal calf serum (Spiro and Bhoyroo, 1974) and has a molecular mass of 48,400 (Graham, 1972), is composed of 78% amino acids, 8.7% sialic acid, 6.3% hexosamine, and 8.3% neutral sugar (Graham, 1972). It has six oligosaccharide side chains per molecule, three of them (of two types) are O-glycosyl-linked to Ser or Thr residues of the protein core, and the other three are identical and N-glycosyl-linked to asparagine (Nilsson et al., 1979). The rat sublingual glycoprotein was prepared by the method of Moschera and Pigman (1975). Its molecular mass is 2.2 × 106 and it is composed of 81% carbohydrates (Moschera and Pigman, 1975). The carbohydrate side-chains are O-glycosyl-linked to Ser or Thr residues of the protein core. The established structure has 9, 10, 12, 13, and 15 sugar residues with NeuNAc[alpha]2,6 linked to Gal and/or GalNAc at the reducing end, GlcNAc groups at nonreducing ends, and a repeating unit, Gal[beta]1->4GlcNAc[beta]1->, in the carbohydrate core structure (Slomiany and Slomiany, 1978). It also contains the Tn determinant (Wu et al., 1995).
Porcine salivary mucin (PSM), bovine submandibular glycoprotein-major (BSM), and armadillo salivary glycoprotein (ASG-A) were purified according to the method of Tettamanti and Pigman (1968) and its modification (Herp et al., 1979, 1988). The products had chemical compositions as similar to those reported previously (Tettamanti and Pigman, 1968; Wu and Pigman, 1977; Herp et al., 1979) and gave a single symmetrical peak in the ultracentrifugal sedimentation velocity analysis (1% in 1 M NaCl solution). Hog gastric mucin #4 (Van Halbeek et al., 1982) was partially purified from porcine stomach mucin (type II, M-2378, Sigma Chemical Co.) by centrifugation at 1.0 × 104 g for 3 h and dialyzed against ddH2O (molecular mass cut off 8.0 × 103) with water changed three times a day for 1 week. Human [alpha]1-acid glycoprotein (Fournet et al., 1978) was purchased from Sigma Chemical Co. (St. Louis, MO).
Desialylation of sialic acid containing glycoproteins was performed by mild acid hydrolysis with 0.01 N HCl at 80°C for 90 min, and dialysis against distilled water for 2 days to remove small fragments (Wu and Pigman, 1977).
Inhibiting sugars
D-Gal, D-Fuc, L-Fuc, D-Man, D-Glc, GalNAc, GlcNAc, melibiose, maltose, sucrose, raffinose, stachyose, methyl[alpha]Gal, methyl[beta]Gal, p-nitrophenyl[alpha]Gal, p-nitrophenyl[beta]Gal, p-nitrophenyl[alpha]GalNAc, p-nitrophenyl[beta]GalNAc, Lac, Gal[alpha]1->4Gal, Gal[alpha]1->3Gal, Gal[alpha]1->3Gal[alpha]->methyl, Gal[beta]1->3GalNAc, Gal[beta]1->3GalNAc[alpha]->benzyl, Gal[beta]1->3GlcNAc, and Gal[beta]1-> 4GlcNAc, phenyl[beta]Gal, L-Ara, Gal[beta]1->3Ara, were purchased from Sigma Chemical Co., St. Louis, MO.
The microtiter plate lectin-enzyme binding assay
The test was performed according to the procedures described by Duk et al. (1994). The volume of each reagent applied to the plate was 50 µl/well, and all incubations, except for coating, were performed at 20°C. The reagents, if not indicated otherwise, were diluted with TBS containing 0.05% Tween 20 (TBS-T). The TBS buffer or 0.15 M NaCl containing 0.05% Tween 20 was used for washing the plates between incubations.
The 96-well microtiter plates (Nunc, MaxiSorp, Vienna, Austria) were coated with untreated or desialylated glycoproteins at the amount of 1 to less than 1 x 10-4 (Figure 1a,b) and 10 to less than 1 x 10-4 (Figure 1c,d) µg per well in 0.05 M carbonate buffer, pH 9.6, overnight at 4°C. After washing the plate, biotinylated lectins were added and incubated for 30 min. The plates were washed to remove unabsorbed lectin and the ExtrAvidin/alkaline phosphatase solution (Sigma, diluted 1:10,000) was added. After 1 h the plates were washed at least four times and incubated with p-nitrophenyl phosphate (Sigma 104 phosphatase substrate 5 mg tablets) in 0.05 M carbonate buffer, pH 9.6, containing 1 mM MgCl2 (1 tablet/5 ml). The absorbance was read at 405 nm in a microtiter plate reader, usually after 2 h incubation with the substrate.
For inhibition studies, the serially diluted inhibitor samples were mixed with an equal volume of lectin solution containing fixed amount of lectin. The control lectin sample was diluted twofold with TBS-T. After 30 min at 20°C, the samples were tested in the binding assay, as described above. The inhibitory activity was estimated from the inhibition curve and is expressed as the amount of inhibitor (nmol/well) giving 50% inhibition of the control lectin binding.
All experiments were done in duplicate or triplicate and the data are mean values of the results. The standard deviation did not exceed 10% and in most experiments was less than 5% of the mean value. The control wells, where coating or addition of biotinylated lectin was omitted, gave low absorbance values (below 0.1, read against the well filled with buffer) and were used as blank. It showed that blocking the wells before lectin addition was not necessary, when Tween 20 was used in TBS.
Acknowledgments
This work was supported by Grants from the Chang-Gung Medical Research Project (CMRP No. 676), Kwei-san, Tao-yuan, Taiwan and the National Science Council (NSC 86-2316-B182-001-BC and 84-2811-B182-001R), the National Health Institutes (DOH 85-HR-316 and DOH 84-HR-209), Department of Health, Taipei, Taiwan.
Abbreviations
PA-IL, Pseudomonas aeruginosa (PA-I) lectin; ELISA, enzyme-linked immunosorbent assay; PBS, 0.02 M phosphate-buffered saline; TBS, 0.05 M Tris-HCl buffered saline, pH 7.4; Gal, D-galactopyranose; Glc, D-glucopyranose; LFuc or Fuc, L-fucopyranose; GalNAc, 2-acetamido-2-deoxy-D-galactopyranose; GlcNAc, 2-acetamido-2-deoxy-D-glucopyranose; gp, glycoprotein. BSM, bovine submandibular gp-major; ASG, armadillo submandibular gp; PSM, porcine salivary gp-major; RSL, rat sublingual gp-major; BN, bird nest glycoprotein, a Gal[alpha]1->4Gal containing glycoprotein. Lectin determinants that are used to classify applied lectins are expressed in bold (Wu and Sugii, 1988 and 1991; Wu et al., 1997a): A(GalNAc[alpha]1->3Gal), Ah (GalNAc[alpha]1->3(LFuc[alpha]1->2)Gal), B(Gal[alpha]1-> 3Gal), T(Gal[beta]1->3GalNAc), I/II (Gal[beta]1->3/4GlcNAc), F(GalNAc[alpha]1->3GalNAc), L(Gal[beta]1->4Glc), Tn(GalNAc[alpha]1-> Ser/Thr), E(Gal[alpha]1->4Gal) (the human blood group Pk active disaccharide which is also part of P1 determinant but this disaccharide is not the key sequence for its reactivity).Structure I. The mucus glycoproteins, the so-called nest-cementing substances, from the salivary gland of Chinese swiftlets (genus Collocalia) are mainly constituted of sialic acid-rich O-glycosylproteins (Wieruszeski et al., 1987). The most complex representatives of the monosialyl fraction from Collocalia mucin are:
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and
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The other compounds identified are partial structures thereof.
Structure II. Proposed representative carbohydrate side chains of blood-group-active glycoproteins, prepared from human ovarian cyst fluid. This structure represents the internal portion of carbohydrate chains to which various human blood group determinants are attached. The four-branched structure (I-IV) shown is the internal portion of the carbohydrate moiety of blood group substances to which the residues responsible for A, B, H, Lea, and Leb activities are attached (Table II). This structure which represents precursor of blood group active glycoproteins (Vicari et al., 1970; Wu et al., 1982; Wu, 1988) can be prepared by Smith degradation of A, B, H active glycoproteins, purified from human ovarian cyst fluids (Howe et al., 1958; Maisonrouge-McAuliffe and Kabat, 1976; Wu et al., 1982; Wu et al., 1984; Mutsaers et al., 1986; Wu, 1988). Numbers in parentheses indicate the sites of attachment for the human blood group A, B, H, Lea, and Leb determinants which are listed in Table II. These determinants as well as the structural units at the nonreducing end are the sources of lectin A/Ah, B, I/II, T, and Tn determinants (Mäkelä, 1957; Wu and Sugii, 1988; Wu et al., 1992). A megalo-saccharide of 24 sugars has not been isolated. However, most of the carbohydrate chains isolated are parts of this structure.
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References
3To whom correspondence should be addressed at: Glyco-immunochemistry Research Lab., Institute of Molecular and Cellular Biology, Chang-Gung Medical College, Kwei-san, Tao-yuan, 333, Taiwan
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