A common epitope of β-lactoglobulin and serum retinol-binding proteins: Elucidation of its core sequence using synthetic peptides

Mokcular hmunology, Vol. 29, No. 4, pp. 51l-516, 1992 Printed in Great Britain.

016L5890/92 $5.00 + 0.00 0 1992 Pergamon Press pie

A COMMON EPITOPE OF /?-LACTOGLOBULIN AND SERUM RETINOL-BINDING PROTEINS: ELUCIDATION OF ITS CORE SEQUENCE USING SYNTHETIC PEPTIDES B.M.M.M.

KUMAR~DDY,AN~ALI

A.

KARANDE

and P. ~DHAKA~

Centre for Reproductive Biology and Molecular Endocrinology Indian Institute of Science, Bangalore-

and Department 012, India

ADIGA* of Biochemistry,

(First received 22 May 1991; accepted in revisedform 19 August 1991) Abstract--&e of the monoclonal antibodies raised against bovine ~-lactoglobulin reacted with human serum retinal binding protein. The finding that this mon~Iona1 antibody also reacted with the serum retinol binding proteins isolated from other animals, suggested that this epitopic conformation is conserved among these proteins. Using ELISA and various synthetic peptides of defined sequence, we show in this paper that the epitope defined by this monoclonal antibody

comprises of the highly conserved core sequence of DTDY present in fi-lactoglobulin and retinol binding proteins.

MATERIALS Ah’D METHODS

INTRODUCTION Both /?-lactoglobulin (BLG) and retinol binding protein (RBP) belong to the superfamily of hydrophobic molecule transporter proteins (Godovac-Zimmermann, 1988). The primary structures of both these proteins are known (Braunitzer et al., 1973; Rask et al., 1979) and their crystal structures show a striking cross hatching of eight strands of anti-parallel p-sheets providing a protective cover for the labile and water-insoluble vitamin A with major differences being confined to surface loops (Papiz et al., 1986). However, the amino acid sequence comparison of both the proteins show only 25-30% homology despite having a close similarity at the level of secondary and tertiary structures (GodovacZimmermann, 1988). It is surmised that the proteins exhibiting more than 40% divergence in their amino acid sequences do not share gross immunological crossreactivities (Prager and Wilson, 1971). The discrete possibility that BLG and RBP could exhibit a limited immunological cross-reactivity dictated by shared amino acid sequences was suggested by our preliminary data that polyclonal antibodies against BLG could bind human RBP in ELISA. In order to decipher the putative common epitopes shared by these two proteins (and possibly by the other members of the superfamily), we used monoclonal antibodies raised against bovine BLG to assess their cross-reactivities, By using one of these MAbs for epitope mapping, the location and the core sequence constituting the common epitope has been elucidated.

Materials

Bovine ~-lactoglobu~n~ human apolipoprotein A II and rabbit antimouse egg-alkaline phosphatase were purchased from the Sigma Chemical Company (U.S.A.). Serum retinol binding proteins were purified to homogeneity from rat, rabbit and human sera as described by Sreekrishna and Cama (1977). Epitope scanning kit was purchased from Cambridge Research Biochemicals, U.K. HC protein was kindly donated by Dr Anders Grubb, Department of Clinical Chemistry, University of Lund, Sweden. Immunization and cell fusion

All the steps involved in the immunization of mice with the antigen, fusion of splenocytes with Sp2/0 myeloma cells and subsequent su~loning were carried out as in Visweswa~ah et al. (1987). Antibody secreting clones were identified by ELISA. Monoclonality was confirmed by subclass isotyping. Cross-reactivity of the BLG MAbs with serum retinol binding proteins

Direct binding assay was used and indi~dual proteins were coated in microtiter plate (lO~g/ml). Following incubation with different MAbs, their binding was quantified by using rabbit antimouse IgG and “‘Ilabelled protein A (Brown et al., 1983), in a solid-phase radioimmunoassay (SPRIA). 3indi~g of the MA6 878 10 to denatured proteins

*Author to whom correspondence should be addressed. Abbreviations: MAb, monoclonal antibody; BLG, plactoglobulin; hRBP, human retinol binding protein; SPRIA, solid-phase radioimmunoassay; ELISA, enzymelinked immunosorbent assay.

To ascertain the type of epitope, i.e. continuous or discontinuous, recognized by the MAb, the reactivities of the MAb towards the native and disulphide reduced carboxymethylated (RCM) proteins were determined. Reduction and alkylation was carried out according to 511

512

B.M.M.M.

KIJMARREDDY~~ ul.

b

a

Konigsberg (1972). These modified proteins were separately immobilized (10 p&/ml) and incubated with the MAb B7BlO. Binding of the MAb was quantified by SPRIA. Inhibition ELISA was carried out by incubation of various concns of native and RCM-BLG with appropriately diluted MAb for 6-8 hr at room temp followed by incubation of these mixtures with immobilized BLG (2.S~g~ml). The binding of the free MAb was determined by antimouse IgG conjugated to alkaline phosphatase (Engvall, 1980).

-

21K

-

18.5 K -

Immunoblotting The proteins were separated by SDS-PAGE and transferred to immobilon-P (Millipore, U.S.A.) membranes (Towbin et al., 1979). After blocking the unoccupied sites with 3% BSA and PBS, the membranes were probed either with the MAb B7BIO or anti BLG antibodies and the binding visualized by autoradiography following treatment with rabbit antimouse IgG and ‘251-labelled protein A. After each step, the membranes were washed repeatedly with 50 mM phosphate buffer (pH 7.2) containing 1 M NaCl, 0.05% Triton X-100, 0.25% gelatin and 5 mM EDTA. Peptide synthesis and epitope identiJcation All the peptides were synthesized on polyethylene pins using Fmoc-amino acid active esters (Geysen et al., 1984), supplied with the Epitope Scanning kit (Cambridge Research Biochemicals, U.K.) according to manufacturer’s instructions. The immunoreactivity of the peptides was assessed by incubating the pins with different MAbs followed by the addition of antimouse IgG conjugated to alkaline phosphatase. 4-Nitrophenyl phosphate was used as phosphatase substrate and A,, was measured in an ELISA reader (BIO-TEK Instruments, Inc, U.S.A., Model EL 307). Other rnet~o~ Native human RBP was subjected to cyanogen bromide cleavage (Gross and Witkop, 1962) and a 11 kDa peptide was isolated and characterized by N-terminal analysis (Chang et al., 1978). This peptide corresponded to the C-terminal 74-182 amino acid residues in the protein. The binding of the MAb B7BlO to this polypeptide was ascertained after covalent coupling to microtiter plates with glutaraldehyde (Tijssen, 1986), and the MAb binding was determined by SPRIA.

iFig. 1. Western blot analysis of (a) hRBP and (b) BLG as probed with anti-BLG antibodies (similar results were obtained with the MAb B7BlO).

recognized by anti-BLG MAbs have been identified (Kaminogawa et al., 1989), the possible existence of epitopes shared by the members of the superfamily have not been considered so far. Having confirmed the immunoiogicai cross-reactivity by Western blotting (Fig. 1) and ELISA (Fig. 2) between BLG and human RBP, using polyclonal anti-BLG antibodies, we considered it worthwhile to identify the common epitopes shared by these two proteins using anti-BLG MAbs. Of the two anti-BLG MAbs investigated, the one, viz. B7BlO (sub-class IgG 2a), that interacted with human serum RBP was selected (Table 1) for further study. The MAb B7BlO exhibited similar affinities for both the proteins in the ELISA (Fig. 3). Since the MAb B7BlO presumably recognizes a common structural feature present in these two proteins, it was of interest to ascertain whether it also cross-reacts with the RBPs isolated from other species since a high degree of sequence homology exists among these RBPs (Sundelin et al., 1985). From Fig. 4, it is clear that the MAb B7BlO is indeed capable of reacting with the serum RBPs purified from rat and rabbit as well, thereby establishing the highly conserved nature of the epitopic conformation recognized by this MAb in these proteins. In order to 2 .O[

RESULTS AND DISCUSSION

Recently elucidated structural homologies among BLG, RBP Apolipoprotein D, HC protein (a-l micro globulin) and BG protein from olfactory epithelium have enabled their classification as members of a new protein superfamily of hydrophobic molecule transporters (Godovac-Zimmermann, 1988). Even though MAbs have previously been raised to serum RBPs (Kumar Reddy et al., 1990; Davila-Bloom et al., 1990) and BLG (Kaminogawa et al., 1987), and two of the tryptic peptide sequences (residues 8-19 and 125-135)

/

1.6

I

’

o--o

BLG

H

hRBP

I

0 0 1.2 c: 0.

. 0

4

.

0.4 j:::::::

I 1:50

,

l

I:500

i 1:5m

i

14nt1serui-n dliutwn

Fig. 2. Binding of varying concns of (1: 50-l : 5000) anti BLG antibodies to BLG and hRBP (10 fig/ml) in ELISA.

Common epitope of j?-lactoglobulin

513

and retinol binding proteins

Table 1. Reactivities of anti fl-lactoglobulin monoclonal antibodies with native and reduced carboxymethylated proteins belonging to superfamily of hydrophobic molecule transporters and the C-terminal peptide of human RBP Bound cpm* Monoclonal H9ElO

Native

Reduced carboxymethylated

BLG hRBP HC protein Apo AI1

65,000

BLG hRBP HC protein Apo AI1 CNBr peptide (hRBP 74-182)

70,000 -

antibodies B7BlO 70,000 69,000 65,000 62,000 35,000

“Input radioactivity, 1 x lo5 cpm. (-) represents the value considered to be non-specific.

investigate

the nature

of the epitope specified by this or discontinuous, the completely unfolded proteins obtained by reduction and carboxymethylation were tested for MAb binding. The positive

MAb, i.e. continuous

data obtained (Table 1) was the first clue that this MAb indeed recognizes a continuous epitope shared by both the proteins. Immunoblot analysis also confirmed the above conclusion (Figs 1 and 4). In attempts to localize the amino acid sequence corresponding to the epitope specified by the MAb B7B10, the reactivity of the MAb was tested with the CNBr-fragment (residues 74-l 82) of human RBP. From the data of Table 1, it is clear that the CNBr fragment harbours the epitope for this MAb. The sequence comparison between this CNBr peptide and BLG revealed a linear stretch of a common sequence DTDY (residues 108-I 11 in human RBP and residues 96-99 of BLG), flanked on one side by hydrophobic amino acids and on the other by charged amino acids (Fig. 5). To delineate the exact amino acid sequence recognized by this MAb, a series of defined decapeptide sequences containing DTDY, at its core and flanked by others as present both in BLG and RBP were syn-

I

I

thesized by solid-phase technique (Geysen et al., 1984) and the propensity of these peptides to bind MAb B7BlO was investigated. It is clear from the Table 2 that the MAb in fact recognizes the DTDY containing sequences and not the others. In order to narrow down the sequence responsible for the MAb binding, a series of overlapping hexapeptides corresponding to the above decapeptides of BLG and RBP were synthesized and tested for the binding of the MAb B7BlO. It is clear that all the hexapeptides containing DTDY sequence (IVDTDY, VDTDYD and DTDYDT of hRBP as well as VLDTDY, LDTDYK and DTDYKK of BLG) exhibited good interaction with the MAb. On the other hand, the other peptides (TDYDTY and DYDTYA of hRBP, TDYKKY and DYKKYL of BLG) were completely negative in this respect (Figs 6a and b). Therefore it is reasonable to conclude that DTDY is the core sequence responsible for the MAb B7BlO binding. As expected (Gnann et al., 1989) the MAb B7B 10 bound the decapeptide to a greater extent than the corresponding hexapeptides as long as DTDY core sequence is retained (Table 2).

1

a

b

C

O-+Z BLG H hRBP

+ Fig. 3. Binding of the MAb B7BlO to varying concns of BLG and hRBP (50 pg-1.5 pgg/ml).

Fig. 4. Western blot analysis of (a) human, (b) rat and (c) rabbit RBP as probed by the MAb B7BlO.

514

B.M.M.M.

KUMARREDDY

hRBP

I

V

D

T

D

Y

D

T

BLG

V

L

D

T

D

Y

K

K

et al.

-_--Y Al I--y IL

PP-14

L

L

D

T

D

Y

D

N

-F L _----

APO-D

I

L

A

T

D

Y

E

N

Y

(Residues 106-115) (Residues 94-103) (Residues 94-103) (Residues 100-109)

Al

I HC

V

V

H

TN

APO-AI1

Q

T

V

T

D

Y

D

Y

G

EIY Al ~_____~ K D L

(Residues 101-110) (Residues

16- 25)

Fig. 5. Sequence homology around DTDY among the members of protein superfamily of hydrophobic molecule transporters. HC protein exhibits a significant homology with BLG in its primary as well as secondary structures (Pervaiz and Brew, 1985). However, B7BlO MAb failed to bind to this protein (Table l), presumably owing to the presence of a different sequence, viz. HTNY (residues 103-106), in the corresponding region (Fig. 5). It may also be noted that the synthetic hexapeptide VHTNYD did not exhibit MAb binding (Table 2) which is in accordance with the lack of MAb recognition of HC protein. It is noteworthy that the other proteins belonging to the superfamily have ATDY in lieu of DTDY sequence (Godovac-Zimmermann, 1988). Similarly human apolipoprotein AI1 (Apo AII) has VTDY in its primary structure (Fig. 5). When tested, apo AI1 (human) failed to recognize the MAb thereby emphasizing the importance of amino terminal aspartic acid in the epitopic conformation (Table 1). Confirmatory evidence stems from studies on different hexapeptides, where the amino terminal aspartic acid is replaced by hydrophobic amino acids as in apolipoprotein D, and apolipoprotein

1

AI1 (Table 2). Direct evidence regarding the importance of the amino terminal aspartic acid of the DTDY sequence comes from the studies using hexapeptides with different replacements. Thus the substitution of aspartic acid with either alanine or valine completely abolished the MAb binding (Figs 6a and b). Of relevance in this context is the report (Julkunen et al., 1988) that the progesterone-dependent placental protein 14 (PP-14) secreted by the human secretory endometrium and decidua exhibits a significant sequence homology (53.4%) with BLG and contains DTDY (residues 96-99) sequence in its primary structure. It is interesting to note that during studies on immunochemical localization of PP14, the MAb B7BlO showed a strong positive reaction with glandular epithelial but not stromal cells particularly during secretory phase of the human endometrium (data not shown). Secondary structure prediction (Chou and Fasman, 1974; Garnier et al., 1978) and the hydrophilicity maps (Hopp and Woods, 1981) reveal that this sequence is hydrophilic, has a propensity to form a b-turn and could be highly antigenic in all the three proteins, viz. BLG, hRBP and PP-14. It is noteworthy that all the known sequences of serum retinol binding proteins and BLG have this conserved sequence, though the biological significance of such a sequence is not known at present (Braunitzer et al., 1973; Sundelin et al., 1985; Erhardt et al., 1989). Employing this type of sequence-specific

Table 2. Reactivity of the MAb B7BlO with different synthetic peptides at varying dilutions A 405 Peptide

Fig. 6. Binding of the mAb B7BlO to the overlapping hexapeptides (a) BLG (residues 94-103) and (b) hRBP (residues 106-l 15). The peptides were synthesized on polyethylene pins and tested for the binding of the antibody in an ELISA.

IVDTDYDTYA (hRBP 106-115) VLDTDYKKYL (BLG 94-103) VFSRDPNGLP (hRBP 131-145) KIVRQRQEEL (hRBP 150-159) VDTDYD (hRBP 107-l 12) LDTDYK (BLG 955100) VHTNYD (HC 102-107) LATDYE (Apo D 101-106) TVTDYG (Apo AI1 human 17-22) -

represents A,, specific. Amino

1:lO

1:lOO

1:lOOO

1.5 1.5

1.26 1.14

0.46 0.45

1.3 1.16

0.6 0.58

-

-

value (0.05-0.09) considered to be nonacids are represented by single letter code.

Common epitope of /I-lactoglobulin

1

I IO

I

I

100

BLG ( pg /we,l

515

may be indicative of subtle differences between BLG and RBP in terms of elicitation of immune response to the DTDY bearing sequence. In summary, using MAb B7BlO for epitope mapping, we have been able to locate and delineate the amino acid sequence specified by the cognate epitope shared by multiple members of a protein superfamily. These studies handsomely illustrate the potential of the MAbs approach to probe the epitopes shared by proteins with as low as 25-30% sequence homology, despite the general belief that proteins having greater than 40% divergence in the amino acid sequence usually show no discernible immunological cross-reactivity (Prager and Wilson, 1971).

)

Fig. 7. Displacement of MAb B7B10 by the native and reduced carboxy-methylated (RCM)-BLG in inhibition ELISA. 100% specific binding corresponds to Aa5 value obtained with MAb alone.

MAbs, it should be possible to locate such immunorecognizable sequences in the three-dimensional structure of the protein if they are surface exposed. Since unlike RCM-BLG, native BLG did not inhibit MAb binding in inhibition ELISA (Fig. 7), it is likely that the DTDY sequence in the native structure is not available for MAb binding. Similar negative results were obtained with the native hRBP (data not shown). Therefore it would appear that this MAb is more specific for denatured BLG and RBP. Contrastingly, when the native proteins were immobilized as in SPRIA (Table l), the MAb binding was significant, presumably due to the partial unfolding of protein upon binding to the plastic surface. Selection of MAbs recognizing denatured molecules when ELISA is used as a method of screening has been reported earlier (Friguet ea af., 1983; Kumar Reddy et al., 1990). B7BlO appears to belong to this category. It is intriguing that though the polyclonal antibodies raised to BLG could recognize hRBP, polyclonal antibodies to RBP did not bind BLG (Fig. 8). Similarly none of the several MAbs raised to chicken RBP (Kumar Reddy et al., 1990) (or) hRBP (Kumar Reddy et al., unpublished results) recognized BLG. On the other hand, we obtained at least three MAbs from the BLG immunized mice, which were similar to B7B10, in terms of their capacity to recognize hRBP. These observations

O--O ELG H

and retinol binding proteins

hRBP

Fig. 8. Binding of varying concns of (1:50-l : 5000) anti-hRBP antibodies to BLG and hRBP (IO tie/ml)I in ELISA. \ I”, ~------

Ac~n~~fedgeme~is-The financial assistance from the Council of Scientific and Industrial Research, New Delhi, is gratefully acknowledged.

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