Vol. 109. No. 3. 1982 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

December 15, 1982 Pages 895-901

CROSS REACTION OF ANTIBODIES AGAINST LIVER GAP JUNCTION PROTEIN (26~) WITH LENS

FIBER JUNCTION PROTEIN (MIP) SUGGESTS STRUCTURAL HOMOLOGY BETWEEN THESE TISSUE

SPECIFIC GENE PRODUCTS

Otto Traub and Klaus Willecke*

Institut fur Zellbiologie, Universitgt Essen, Hufelandstr. 55, 43 Essen 1, Federal Republic of Germany

Received November 5, 1982

SUMMARY : The recently characterized antiserum against the sodium dodecyl sulfate-denatured 26K protein from mouse liver gap junctions (Traub, O., Janssen-Timmen, U., Driige, P.M., Dermietzel, R., and Willecke, K., (1982) J. Cell. Biochem. 19, 27-44) was affinity purified and its reactivity with the junction protein (MIP) from mouse or bovine lens fiber tissue was investi- gated using the immunoblot technique. A weak reaction of MIP with anti-26K antiserum was clearly noticeable. The results of control experiments exclude nonspecific binding of IgG molecules to MIP. The specific, although weak, reaction of anti-26K antiserum with MIP hints that both gap junction proteins, liver 26K and lens fiber MIP, share some structural homology which had not previously been recognized. We suggest that liver and lens junction proteins may be related products of a gene family which may also encompass gap junction proteins expressed in other mammalian tissues.

Gap junctions are built up of membrane proteins which form channels directly

connecting contiguous cells in tissues or in culture (1). Major gap junction

polypeptides isolated from livers of mouse and rat were found to have apparent

molecular weights between 26~ and 28K, respectively (2-4). Recently the N-term-

inal 52 amino acids of the rat liver protein (28K) were sequenced (5). The

purification of gap junction protein from calf lens has also been described (6).

This protein is likely to be identical to the "main intrinsic polypeptide" (MIP)

from bovine lens (7). Several authors had reported that rabbit anti-bovine MIP

antisera do not cross react with other tissues including bovine liver, or with

the major gap junction protein from rat liver (7-11). Nicholson et al. (12)

found no homology in the amino acid sequence near the N-terminus of the liver

and lens junction protein. Hertzberg et al. (11) concluded from their analysis

*To whom correspondence should be addressed. Present address: Department of Biological Sciences, Stanford University, Stanford, California 94305, USA.

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of the proteolytic peptide pattern that both proteins were different. Recently

Zampighi et al. (13) presented evidence that lens gap junctions have a tetra-

merit subunit structure whereas liver gap junctions appear to be hexameric. We

have recently characterized a rabbit antiserum which specifically recognized

the 26K protein in denatured (14) and native (15) mouse liver plasma membranes.

In this communication we investigated the cross reactivity of affinity purified

anti-liver 26K antiserum with mouse and bovine lens junction protein (MIP).

MATERIALS AND METHODS:

Gap junction plaques from mouse liver were isolated according to Henderson et al. (2,14) and from mouse or bovine lens according to Goodenough (6). In addition, MIP was isolated from bovine or mouse lens using the method of Bouman et al. (16). Bovine MIP, prepared by Dr. R.M. Broekhuyse (Nijmegen), co-migrated electrophoretically with our MIP preparations.

Rabbit anti-liver 26K antiserum previously characterized in this laboratory (14,15) was affinity purified on electrophoretically separated mouse liver 26K

protein using diazobenzoxymethyl (DBM) paper (14) according to the method of Olmsted (17). Rabbit anti-bovine lens MIP antiserum was kindly given to us by Dr. R.M. Broekhuyse (Nijmegen). Affinity purified rabbit anti-chick gizzard vinculin IgG (1 mg/ml) (18) was a gift of Dr. B. Jockusch (Bielefeld) and affinity purified rabbit anti-chick type I collagen antiserum (0.5 mg/ml) (19) was donated to us by Dr. K. von der Mark (Martinsried). Reference samples of bovine lens crystallins were given to us by Dr. R. Vornhagen (Bonn). All preparations of gap junction protein from liver or lens were analyzed after electrophoresis on polyacrylamide gels (12.5%) in the presence of sodium dodecyl sulfate (SDS) (20). The proteins were blotted by capillary action (21,22) or by electrophoretic transfer (23) onto nitrocellulose paper. Antigen-antibody complexes were labeled with [ 12511 protein A (Amersham-Buchler, specific activity: 30 mCi/mg (14)) or with goat F(ab)2 anti-rabbit [125I] IgG (NEN, specific activity: 2-10 @i/pg). The experimental conditions were analogous to those for [12511 protein A (14). In later experiments the antigen-antibody complexes were detected by horseradish peroxidase (Boehringer) coupled (24) to goat anti-rabbit IgG (Miles) fused at 50 fold dilution). The staining reaction was carried out with the chromogen 3,3'-diamino-benzidine tetrahydrochloride (Schuchardt) (25).

RESULTS:

The purified proteins from mouse or bovine lens were electrophoresed on

SDS polyacrylamide gels, stained with Coomassie blue and the apparent molecular

weights were compared (Figure 1). We estimated an apparent molecular weight of

25K for the lens junction protein (MIP) in accordance with recent results of

Hertzberg et al. (11) but in contrast to earlier reports by Goodenough (6) and

Broekhuyse et al. (7).

The protein preparations represented in Figure 1 were transferred to nitro-

cellulose paper and their reactions with different antisera were investigated.

Figure 2 (lane Ah) illustrates that the anti-liver 26K antiserum reacted weakly

Vol. 109. No. 3, 1982 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

ABCD Start

68K )I

45K -

29K- 26K )I rl-

14K * ,

A -a b

start - fr’

7, ’

26K -b-

B C ab ab ' 1 ,

i

Figure 1. Comparison by electrophoresis on SDS polyacrylamide gel of gap junction protein from liver and lens fiber tissue. The following protein preparations were applied onto the gel: Lane A: gap junction plaques (3 pgl from mouse liver; lane B: gap junction protein (6 ug) from bovine lens prepared according to (6); lane C: main intrinsic polypeptide (MIP) (10 ug) from bovine lens and mouse lens (lane D, 10 pg), both prepared according to (7,16). All proteins were stained with Coomassie blue. The following proteins were used for reference purposes: bovine serum albumin 168K), ovalbumin (45Kl. carbonic anhydrase (29K), chyotrypsinogen (26~), and ribonuclease (14K).

Figure 2. Immunoblot of gap junction protein from mouse liver and bovine lens fiber tissue usinq different antisera. The following proteins were separ- ated by SDS polyacrylamide gel electrophoresis. Lanes A: gap junction protein (1 ug) from mouse liver and Lanes B and C: gap junction protein (5 ug) from bovine lens. After transfer to nitrocellulose paper, strip A was incubated with anti- 26K antiserum (1:lOO diluted), strip B with preimmune serum cl:100 diluted) and strip C with anti-MIP antiserum (1:lOOO diluted). The antigen-antibody complexes were detected by the horseradish peroxidase method described under Materials and Methods.

with lens 25~ protein (MIP). Preimmune serum, however, also reacted with lens

25K protein (MIP) (lane Bb) (but not with liver 26K protein (lane Ba)) to

almost the same extent as anti-liver 26~ antiserum. No reaction of anti-MIP

antiserum with the liver 26K protein was found (lane Ca). Anti-MIP antiserum

reacted strongly (as expected) with the 25K lens protein (lane Cb) purified

according to Goodenough (6) or Bouman et al. (15). In Figure 2 the antigen-

antibody reaction was detected by indirect staining after incubation with

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Vol. 109, No. 3. 1982 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

ABCD StOH

fC-- f

@ . .

Figure 3. Immunoblot of liver and lens fiber junction protein using affinity Purified anti-26K antiserum. Lanes A and B were incubated with affinity purified anti-26K antiserum after electrophoresis and transfer to nitrocellulose paper of mouse liver gap junction protein (1 ug, lane A) and bovine lens junction protein (2 ug, lanes B,C,D). The paper strip of lane C was incubated with rabbit anti-MIP antiserum (1:lOOO fold diluted) and lane D was incubated with rabbit anti-vinculin IgG (1:lOOO fold diluted). Liver and lens junction protein tend to form aggregates of higher molecular weight (2,16).

anti-rabbit IgG and horseradish peroxidase. The same result was found when we

analyzed the position of the antigen-antibody complexes after immunoblot with

[1251] protein A or with goat anti-rabbit [125~] F(abj2 followed by autoradio-

graphs - In a series of control experiments we did not observe any binding of

the indicator molecules (horseradish peroxidase, protein A, or goat anti-rabbit

F(ab)2) at the same concentrations used before to the liver 26K or lens 25K

protein (MIP) (results not shown).

In order to analyze whether or not the weak cross reaction of anti-26K

antiserum with MIP (Figure 2, lane Ab) was due to specific binding we purified

the anti-26K antiserum by affinity chromatography. Following Olmsted's

procedure (17),the anti-26K antibodies bound to the 26K protein band on DBM

paper (10 cm2) were eluted at acid pH and then neutralized. Figure 3, lane B,

clearly indicates that affinity purified anti-26K antibodies bind to lens fiber

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junction protein (MIP). The concentration of affinity purified anti-26K anti-

bodies used for this reaction was estimated after SDS-polyacrylamide electro-

phoreses, silver staining, and densitometric evaluation of the protein bands

to be about 0.3 ug/ml (total volume: 2ml). In lane A of Figure 3 the much

stronger reaction of affinity purified anti-26K antibodies with liver 26K

protein is illustrated. MIP does not bind nonspecifically to rabbit IgG at the

concentrations used before. This is shown in lane D of Figure 3. No reaction

is seen when MIP after electrophoresis and transfer to nitrocellulose paper was

incubated with affinity purified anti-vinculin IgG at the concentration of 1 ug/m.

or with affinity purified rabbit anti-collagen type I antiserum at 0.5 ug/ml

(not shown). Thus we conclude that anti-26K antiserum must contain antibodies

which specifically recognize lens fiber protein (MIP), although the reaction is

probably only about l-2% as strong as with liver 26K protein (estimated by

densitometric evaluation of the corresponding band on autoradiographs). We

found no cross reaction of a, 6, or y crystallin, isolated from bovine lens,

with anti-26K antiserum. Thus the reaction of affinity purified anti-26K

antiserum with MIP cannot be due to contamination of the MIP preparation with

crystallin proteins. Furthermore, MIP when present in whole tissue homogenates

from mouse or bovine lens, reacted with anti-26K antiserum identically as after

purification. This result excludes that the reaction of MIP with anti-26K

antiserum is due to an artifact introduced during purification of MIP.

DISCUSSION:

We have shown in this communication that affinity purified anti-liver 26K

antiserum reacts specifically with lens junction protein (MIP), although to a

much lower extent than with liver 26K gap junction protein. We do not know

why non-immunized rabbits appear to have a low titer of IgG molecules reacting

with MIP. One may argue that anti-26K antiserum which was raised after injection

of SDS denatured mouse liver 26K protein into rabbits contains antibodies which

recognize SDS molecules bound to protein and may therefore react with SDS

molecules bound to lens protein (MIP). We think this interpretation unlikely

since anti-26K antiserum reacts specifically with liver 26K protein when proteins

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vol. 109, No. 3, 1982 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

of total liver membranes were subjected to electrophoresis and immunoblot (14).

Since many liver membrane proteins can be expected to bind SDS one should see

a background of unspecific binding of anti-26K antibodies to many liver membrane

proteins. Our results suggest that both gap junction proteins, liver 26K and

lens MIP, share some structural homology. Carbohydrates are unlikely to be

involved since the liver 26K protein (3) and lens MIP (7) do not appear to be

glycosylated. The fact that anti-MIP antibodies do not appear to react with

liver 26~ protein, as observed in this paper and reported from other laboratories

(7-ll), could be due to different methods of antigen preparation or conditions

of immunization. Furthermore, the postulated region of homology in the structure

of liver and lens junction protein may elicit a different immunogic response

depending on the different immunogenic sites of both proteins. Although liver 26K

protein and lens fiber MIP have similar molecular weight, morphology and basic

function (i.e., to permit the transfer of small molecular weight compounds between

contiguous cells) several investigators have pointed out the differences between

these proteins (10-12). Hertzberg et al. (11) predicted that antibodies to the

liver gap junction polypeptide would not cross react with the lens fiber junction

polypeptide. The results of this communication contradict this prediction.

Furthermore in two cases reported in the literature (26,27) homology between

proteins remained undetected by peptide mapping but was later found upon amino

acid sequencing. Only further sequence analysis of the liver and lens junction

proteins will determine unambiguously whether they may be related. We neverthe-

less suggest that in the mammalian genome a family of genes for gap junction

proteins may exist which stems from a common ancestral DNA sequence. Different

gap junction genes may have evolved due to selective pressure during evolution

and are now expressed in a tissue specific manner such as are the liver and lens

junction proteins. In future studies this hypothesis can be checked by molecular

comparison of gap junction proteins from different tissues and by analysis of

the corresponding genes.

ACKNOWLEDGMENTS:

We thank Drs. R.M. Broekhuyse (Nijmegen), B. Jokusch (Bielefeld), K. von der Mark (Martinsried) for antisera and Dr. R. Vornhagen (Bonn) for samples of

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purified crystallins as well as for discussions of our results. The technical assistance of Beate Karow is gratefully acknowledged. This work was supported by grants of the Ministerium fur Wissenschaft und Forschung, Dusseldorf, and (in part) by the Deutsche Forschungsgemeinschaft (SFB 102).

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