Báo cáo khoa học: A (1fi3)-b-D-glucan recognition protein from the sponge Suberites domuncula Mediated activation of fibrinogen-like protein and epidermal growth factor gene expression

A (1→3)-β-D-glucan recognition protein from the sponge Suberites domuncula

Tác giả: Sanja Perović-Ottstadt, Teresa Adell, Peter Proksch, Matthias Wiens, Michael Korzhev, Vera Gamulin, Isabel M. Müller and Werner E. G. Müller

Lĩnh vực: Sinh học phân tử, Hóa sinh

Nội dung tài liệu: Nghiên cứu này tập trung vào việc khám phá cơ chế nhận biết nấm của bọt biển Suberites domuncula. Các nhà khoa học đã xác định được một protein thụ thể trên bề mặt tế bào có khả năng nhận biết các phân tử (1→3)-β-D-glucans, một thành phần cấu tạo quan trọng trong thành tế bào của nấm. Việc phát hiện protein này đã dẫn đến phân tích trình tự DNA bổ sung (cDNA) và phát hiện ra rằng gen mã hóa cho protein 45 kDa này được điều hòa tăng cường sau khi tiếp xúc với carbohydrate. Đồng thời, hai gen khác cũng cho thấy mức độ biểu hiện ổn định tăng lên, bao gồm một gen mã hóa protein giống fibrinogen và một gen mã hóa tiền chất yếu tố tăng trưởng biểu bì. Nghiên cứu cũng chỉ ra rằng một con đường truyền tín hiệu tyrosine kinase được khởi phát khi tiếp xúc với D-glucan, và khả năng phosphoryl hóa của nó có thể bị ngăn chặn bởi aeroplysinin. Kết quả này cho thấy bọt biển sở hữu một cơ chế phân tử để nhận biết nấm thông qua carbohydrate D-glucan trên bề mặt của chúng.

Mục lục chi tiết:

• A (1→3)-β-D-glucan recognition protein from the sponge Suberites domuncula
• Mediated activation of fibrinogen-like protein and epidermal growth factor gene expression
• Sponges (phylum Porifera) live in a symbiotic relationship with microorganisms, primarily bacteria.
• Keywords: D-glucan binding protein; epidermal growth factor; fungi; sponges; symbiosis.
• Sponges (phylum Porifera) are, among all metazoan taxa, those animals which contain the widest range of specific and very effective bioactive compounds [1,2].
• Correspondence to W. E. G. Müller, Institut für Physiologische Chemie, Abteilung Angewandte Molekularbiologie, Universität, Duesbergweg 6, 55099 Mainz, Germany.
• Abbreviations: EGF, epidermal growth factor; LPS, lipopolysaccharide; MAPK, mitogen-actived protein kinase; PoAb, polyclonal antibody.
• Note: This article is dedicated to Professor Zeeck (University of Göttingen) on the occasion of his 65th birthday.
• Note: The CDNA sequences from Suberites domuncula have been deposited in EMBL/GenBank as follows: the (1→3)-β-D-glucan-binding protein (GLUBPp_SUBDO) under the accession number AJ606470, the fibrinogen-like molecule (FIBI_SUBDO) under the accession number AJ606471, and the epidermal growth factor precursor (EGFI-PREC_SUBDO) under the accession number AJ606469.
• (Received 23 January 2004, revised 9 March 2004, accepted 22 March 2004)
• assumed that most of these secondary metabolites are produced by symbiotic microorganisms which are harbored by the sponges [3].
• Activation of sponge cells by (1→3)-β-D-glucan (Eur. J. Biochem. 271)
• which have been isolated from cell walls of plants, but also from bacteria and fungi [8].
• Materials and methods
• Chemicals and enzymes
• Sponges
• Exposure of tissue samples from S. domuncula to curdlan and Western blotting
• Ligand-binding blot
• Isolation of cDNA for the (1→3)-β-D-glucan-binding protein
• cDNA encoding the fibrinogen-like protein
• cDNA encoding the putative EGF-like precursor, EGFI-PREC_SUBDO
• Sequence analysis
• Recombinant EGF precursor and production of antibodies
• Western blotting of EGF
• RNA preparation and Northern blot analysis
• In situ localization studies
• Results
• Effect of incubation with curdlan on the phosphorylation of tyrosine in sponge tissue
• Fig. 1. Phosphorylation of a 32 kDa protein after incubation of sponge tissue with curdlan.
• Detection of glucan-binding activity in extracts from S. domuncula
• Cloning of cDNA encoding the S. domuncula (1→3)-β-D-glucan-binding protein
• Fig. 2. Detection of a glucan-binding protein in extracts from Suberites domuncula.
• (1→3)-β-D-glucan-binding proteins and was therefore termed GLUBPp_SUBDO.
• Phylogenetic analysis.
• cDNA encoding the fibrinogen-like protein
• Fig. 3. The Suberites domuncula potential beta-1,3-glucan-binding protein (GLUBPp_SUBDO).
• Fig. 4. Suberites domuncula fibrinogen-like protein.
• cDNA of a potential EGF precursor
• Fig. 5. The putative epidermal growth factor (EGF) precursor, EGFI-PREC_SUBDO, from Suberites domuncula.
• Phylogenetic analysis.
• Upregulation of gene expression for the described genes in response to curdlan: Northern blotting
• Fig. 6. Steady-state expression of the SDGLUBP gene [(1→3)-β-D-glucan-binding protein], the SDFIBI gene (fibrinogen-like protein) and the SDEGFI-PREC gene (epidermal growth factor EGF precursor) in tissue from Suberites domuncula after exposure to 10 µg·mL⁻¹ curdlan.
• Identification of cells expressing glucan-binding protein and fibrinogen
• Fig. 7. Spatial expression pattern of (1→3)-β-D-glucan-binding protein and fibrinogen in sections of Suberites domuncula.
• Level of low-molecular-weight EGF in tissue after treatment with curdlan
• Fig. 8. Level of epidermal growth factor (EGF) in tissue after treatment with curdlan.
• Discussion
• sponge S. domuncula.
• Parallel experiments were performed to determine the expression of the SDFIBI gene (fibrinogen-like protein).
• In view of the above finding, that curdlan causes phosphorylation of Tyr residue(s) in polypeptides of S. domuncula, which is prevented by aeroplysinin, it seemed necessary to determine whether this protein tyrosine kinase inhibitor also has an effect on the pronounced increase of expression of the three genes under study.
• Taken together, the data presented here demonstrate that sponges, in this study using S. domuncula as the model, comprise a (1→3)-β-D-glucan-binding protein, which bind to (1-3)-β-D-glucans, e.g. curdlan or laminarin.
• Acknowledgements
• References