Suppression of allograft rejection in the sponge Suberites domuncula by FK506 and expression of genes encoding FK506-binding proteins in allografts
Werner E. G. Müller1,*,
Renate Steffen1,
Bernd Lorenz1,
Renato Batel3,
Michael Kruse2,
Anatoli Krasko1,
Isabel M. Müller1 and
Heinz C. Schröder1
1 Institut für Physiologische Chemie, Abteilung Angewandte Molekularbiologie, Universität, Duesbergweg 6, D-55099 Mainz, Germany,
2 Max-Planck-Institut für Züchtungsforschung, Carl-von-Linné-Weg 10, D-50829 Köln, Germany and
3 Center for Marine Research, ‘Ruder Boskovic’ Institute, HR-52210 Rovinj, Croatia
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Fig. 1. Effect of FK506 on allograft rejection/non-fusion and autograft fusion of transplants from Suberites domuncula. (A) A specimen of S. domuncula also showing the hermit crab Paguristes oculatus. (B) A red and a blue specimen. (C) Graft tied together with a fibre. (D) Beaker in which two grafts are incubated in sea water, as described in Materials and methods. (E–J) In each panel, two grafts are shown that had been incubated in parallel either without (minus; E,G,I) or in the presence of FK506 (plus FK506; F,H,J); the allograft (al) is shown on the left and the autograft (au) on the right. Three different concentrations of the drug were used: 2000ng ml-1 (F), 200ng ml-1 (H) and 20ng ml-1 (J). In I, the fusion zone (fz) in one autograft and the gap (g) in the allograft are marked (arrowheads). E–J were taken 5 days after grafting. Scale bars, 10 mm.
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Fig. 2. Putative FK506-binding proteins FKB1_SUBDO and FKB2_SUBDO. (A) Alignment of the deduced sponge sequences, (FKB1_SUBDO and FKB2_SUBDO) with the human FK506-binding protein (FKB_HOMO; GenBank accession number NP_004107). Residues conserved (identical or similar with respect to their physico-chemical properties) in all sequences are shown in white on black and those in at least two sequences in black on gray. The locations of the two FKBP-type peptidyl-prolyl cis–trans isomerase signatures 1 and 2 (FKBP-1 and FKBP-2) are marked. In addition, the transmembrane helix (TM) is indicated in FKB2_SUBDO. (B) Rooted phylogenetic tree built by two listed sequences (FKB1_SUBDO and FKB_HOMO; Homo) as well as by the related sequences from Manduca sexta (Manduca; AAF16717), Xenopus laevis (Xenopus; O42123), Saccharomyces cerevisiae (Saccharomyces; NP_014264) and Arabidopsis thaliana (Arabidopsis; S72485). The last sequence was used as an outgroup. Scale bar indicates an evolutionary distance of 0.1 amino acid substitutions per position in the sequence.
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Fig. 3. Effects of FK506 on the incorporation of [3H]deoxythymidine into the acid-insoluble fraction from cells of Suberites domuncula. Single cells were incubated in sea water for 2 days and subsequently exposed to different concentrations of FK506 for 24h, as described in Materials and methods. The acid-insoluble fraction was examined and the incorporation of 3H correlated with the protein content of the sample. Values are means ± S.D. of eight experiments.
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Fig. 4. Microscopic analysis of auto- and allografts in the absence (minus; A–D) or presence of FK506 (20ng ml-1; E–H). Grafts 1 day (A,C,E,G) or 5 days (B,D,F,H) after the transplantation are shown. The respective auto- (A,B,E,F) and allografts (C,D,G,H) are also indicated on the top of the figures. The zones between the grafts are marked with filled circles. Scale bar, 1 mm.
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Fig. 5. Transcripts for the two FK506-binding proteins FKB1_SUBDO and FKB2_SUBDO from Suberites domuncula, identified using northern blotting. (A) Determination of the size of the two transcripts, SUBDOFKB1 (FKB1; 0.62kb) and SUBDOFKB2 (FKB2; 0.79kb); the positions of size markers (kb) are given on the left. (B) RNA from transplants was obtained as described in Materials and methods. (i) Autografts (not treated with FK506), (ii) allografts (untreated) and (iii) allografts (treated with 20ng ml-1 of FK506) were taken, RNA was extracted, and northern blots were performed. RNA was extracted from the graft zones, 0 (lane a), 10 (lane b) or 12h (lane c) after the start of the transplantation experiments. The intensities of the bands corresponding to SUBDOFKB1 (FKB1) and SUBDOFKB2 (FKB2) were standardized against those seen with ß-tubulin, as described in Materials and methods. (iv) In parallel, RNA from allografts (treated with 20ng ml-1 FK506) were size-separated and analyzed with the S. domuncula ß-tubulin probe SDBTUB.
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© The Company of Biologists Ltd 2001
