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doi: 10.1242/jcs.01083

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Matrix-mediated canal formation in primmorphs from the sponge Suberites domuncula involves the expression of a CD36 receptor-ligand system

Werner E. G. Müller^1,*, Narsinh L. Thakur¹, Hiroshi Ushijima², Archana N. Thakur¹, Anatoli Krasko¹, Gaël Le Pennec¹, Madhavi M. Indap³, Sanja Perovi-Ottstadt¹, Heinz C. Schröder¹, Gerhard Lang⁴ and Gerhard Bringmann⁴

¹ Institut für Physiologische Chemie und Pathobiochemie, Abteilung Angewandte Molekularbiologie, Johannes Gutenberg-Universität M ainz, Duesbergweg 6, 55099 Mainz, Germany
² Institute of International Health, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-Ku, Tokyo 113-0033, Japan
³ Department of Zoology, D. G. Ruparel College, Mahim, Mumbai 400016, India
⁴ Institut für Organische Chemie, Universität Würzburg, Am Hubland, 97074 Würzburg, Germany

View larger version (66K): [in a new window]   Fig. 1. Formation of canal-like structures in primmorphs from S. domuncula. (A) Dissociated cells were obtained and (B) transferred to seawater-based medium; after 3 days aggregates with a rough surface were formed. (C) After a prolongation of the incubation for 6 days round primmorphs with a smooth surface (surrounded by choanocytes) were formed. (D) A subsequent transfer of the primmorphs onto galectin-coated culture dishes resulted, after six days (total incubation period of 12 days), in the formation of canal-like structures. (E) A higher magnification of the structures. (F) A primmorph that was cultured for the complete incubation period of 12 days in uncoated culture dishes; these structures remained in the non-attached state and were roughly spherical. Original magnifications: (A) x50; (B,C,F) x10; (D) x5; (E) x50.

View larger version (59K): [in a new window]   Fig. 2. S. domuncula CD36/LIMPII receptor. (A) The deduced S. domuncula protein (CD36/LIMPII_SD) is aligned with the human lysosomal integral membrane protein II (LIMPII) (LIMPII_HUMAN; NP_005497.1) (Calvo et al., 1995), and the human CD36 platelet glycoprotein IV (CD36_HUMAN; P16671) (Oquendo et al., 1989). Amino acids, similar among all sequences, are in black boxes and those conserved in at least two sequences are in shaded boxes. The amino acid residues, characteristic for conserved domain `block A' ({-A-}) and the transmembrane region ([TM]) as well as those which represent the internal potential N-linked glycosylation sites, are marked. (B) Phylogenetic tree constructed from the above mentioned sponge sequence and the two human sequences together with the epithelial membrane protein CG2727-PA from Drosophila melanogaster (EMP-CG_DROME; NP_523859.2), the CD36 family member from Caenorhabditis elegans (CD36_CAEEL; NP_499625.1), Spo71p sequence from Saccharomyces cerevisiae (Spo71p_YEAST; NP_010389.1) (Jacq et al., 1997) and the fructose-bisphosphatase precursor protein At3g54050.1 from Arabidopsis thaliana (FBPPP_ARATH; NP_190973.1). After alignment the tree was built and rooted using the plant sequence as an outgroup. Scale bar indicates an evolutionary distance of 0.1 amino acid substitutions per position in the sequence. (C) Comparison of the S. domuncula CD36/LIMPII receptor molecule with the related human sequences: the lysosomal integral membrane protein II (LIMPII_HUMAN), membrane glycoprotein CLA-1 protein (CLA-1_HUMAN; A48528) (Calvo et al., 1993) and CD36 (CD36_HUMAN). A phylogenetic cladogram (slanted), was constructed and, after alignment, rooted with the sponge CD36/LIMPII receptor. The analysis was performed by neighbor-joining as described under Materials and Methods. The numbers at the nodes are an indication of the level of confidence–given as a percentage–for the branches as determined by bootstrap analysis [1000 bootstrap replicates]. Note: The cDNA sequences from Suberites domuncula have been deposited in EMBL/GenBank databases; cDNA for the CD36/LIMPII receptor is under the accession number AJ558195.

View larger version (23K): [in a new window]   Fig. 3. Alignment of the S. domuncula peptide fragment (ADAMTS_SD), deduced from the EST SDADAMTS, with the human ADAMTS-9 precursor, a disintegrin and metalloproteinase with thrombospondin motifs 9 (ADAMTS-9_HUMAN, Q9P2N4) (Clark et al., 2000). The numbers at the human sequence refer to the complete sequence. The sponge polypeptide consists of the CSVTCG peptide domain that bind to the CD36 receptors (CD36BD). The borders of the sequence that were used for the preparation of the recombinant protein are given {-expression-}.

View larger version (51K): [in a new window]   Fig. 4. Expression of a part of the S. domuncula SDADAMTS sequence including the CD36 binding motif. As outlined under Materials and Methods the fragment was subcloned into the bacterial expression vector pET41a and expression was performed in E. coli BL21 using IPTG. Lysates from non-induced (–IPTG; lane a) as well as from induced bacterial cultures (+IPTG; lane b) were prepared and analyzed using 15% polyacrylamide gel containing NaDodSO4; the gel was stained with Coomassie Brilliant Blue. The protein extract containing the recombinant SDADAMTS was purified/enriched by affinity chromatography (lane c).

View larger version (45K): [in a new window]   Fig. 5. Expression of the genes encoding the CD36/LIMPII receptor, galectin as well as ADAMTS in primmorphs in response to different cultivation matrices. (A) The dissociated cells (time 0) were transferred into a seawater-based medium and cultured in uncoated dishes for 6 days (free). Then the primmorphs were transferred onto galectin-coated dishes for either 3 days (total period of incubation: 9 days) or 6 (12) days during which time they attached (attached: arrow). In a parallel series of experiments the primmorphs remained in uncoated dishes where they remained in the free state for 6 (12) days (free). Furthermore, the primmorphs that had been cultured on galectin-coated dishes were co-incubated for 6 (12) days with 10 µg/ml of the recombinant SDADAMTS peptide, containing the CSVTCG motif [+TSP]. RNA was isolated from the respective primmorphs and the same amount of RNA was loaded onto the gels and size separated. After blot transfer the filters were probed with the CD36/LIMPII receptor cDNA. (B) In parallel, the blot was probed with the galectin cDNA. (C) Effect of MTN on primmorphs that had been transferred to galectin-coated dishes for 3 (9) days. Either no compound was added to the cultures, or MTN was added at a concentration of 0.03 or 0.01 µg/ml to the assays for this incubation period. The blot was developed with the CD36/LIMPII receptor cDNA. (D) In parallel, the blot was incubated with the SDADAMTS probe. (E) As a control to show the interaction between the recombinant SDADAMTS peptide and the CD36/LIMPII receptor, this peptide was pre-incubated with equal concentrations of the recombinant CD36/LIMPII receptor polypeptide as described under Materials and Methods (+TSP/rCD36). The primmorphs were incubated on galectin-coated dishes for 6 (total incubation period of 12) days either in the absence of any recombinant polypeptide, or in the presence of 10 µg/ml recombinant SDADAMTS (+TSP), or in the presence of 10 µg/ml recombinant SDADAMTS together with 10 µg/ml recombinant CD36/LIMPII receptor (+TSP/rCD36). The relative degree of expression was correlated with that seen at the related control time point (day 0 or day 6, as indicated); this value is set to 1.

View larger version (167K): [in a new window]   Fig. 6. In situ localization of cells expressing the CD36/LIMPII receptor in tissue of S. domuncula. Cryosections of tissue were hybridized with DIG-labeled SDCD36/LIMPII. Subsequently, the specimens were incubated with anti-digoxigenin/alkaline phosphatase and the signals were detected with NBT/X-phosphate as described under Materials and Methods. (A,C) hybridization with antisense SDCD36/LIMPII; (B,D) hybridization with sense SDCD36/LIMPII. Canals (c) of the aquiferous system within the mesohyl (m) are shown. The canals are lined up by an epithelial layer formed from pinacocytes, which are SDCD36/LIMPII-positive cells. Magnifications: (A,B) x50; (C,D) x100.

View larger version (10K): [in a new window]   Fig. 7. Structure of 2-methylthio-1,4-naphthoquinone (MTN).

View larger version (42K): [in a new window]   Fig. 8. Effect of MTN on vascularization in chick embryos. (A) Control chick CAM after incubation with an untreated agar disc for 48 hours. (B) CAM incubated for the same period with 0.25 ng MTN/disc and (C) with 1 ng MTN/disc. A distinct disorganization of the vessel formation, avascular zone, is seen at the locations where the discs were placed (d; arrowheads). Magnifications: x10.

View larger version (165K): [in a new window]   Fig. 9. Modulation of CD36/LIMPII receptor gene expression in primmorphs; analysis was performed by in situ hybridization. (A) Primmorphs were cultured for 6 plus 3 days in uncoated culture dishes; then the in situ analysis was performed. (B) Primmorphs, cultured for 6 days in uncoated dishes and subsequently for 3 days on the galectin matrix. (C) Higher magnification of an area in primmorphs, grown on galectin. An intense staining of the cells, especially around the canal-like structures (c) is seen. (D,E) Primmorphs that had been cultured on galectin but in addition either in the presence of 10 µg/ml of the recombinant SDADAMTS (D), or 0.03 µg/ml of MTN (E). After the 3 days incubation the primmorphs were analyzed by in situ hybridization with the antisense probe SDCD36/LIMPII. Magnifications: (A,B) x5; (C-E) x15.