Báo cáo khoa học: ˚ cDNA cloning and 1.75 A crystal structure determination of PPL2, an endochitinase and N-acetylglucosaminebinding hemagglutinin from Parkia platycephala seeds

CDNA cloning and 1.75 Å crystal structure determination of PPL2, an endochitinase and N-acetylglucosamine-binding hemagglutinin from Parkia platycephala seeds

Tác giả: Benildo S. Cavada, Frederico B. B. Moreno, Bruno A. M. da Rocha, Walter F. de Azevedo Jr, Rolando E. R. Castellón, Georg V. Goersch, Celso S. Nagano, Emmanuel P. de Souza, Kyria S. Nascimento, Gandhi Radis-Baptista, Plínio Delatorre, Yves Leroy, Marcos H. Toyama, Vicente P. T. Pinto, Alexandre H. Sampaio, Domingo Barettino, Henri Debray, Juan J. Calvete, and Libia Sanz

Lĩnh vực: Biochemistry, Molecular Biology

Nội dung tài liệu: Nghiên cứu này trình bày việc xác định trình tự amino acid đầy đủ của Parkia platycephala lectin 2 (PPL2) thông qua kỹ thuật cDNA cloning và xác định cấu trúc tinh thể 3D ở độ phân giải 1.75 Å. PPL2 được phân lập từ hạt của cây Parkia platycephala và cho thấy hoạt tính ngưng kết hồng cầu thỏ, hoạt tính này bị ức chế đặc hiệu bởi N-acetylglucosamine. Protein này cũng có khả năng thủy phân các liên kết glycosidic β(1-4) trong chitin. Cấu trúc tinh thể cho thấy PPL2 có cấu trúc tương đồng với các endochitinase thuộc họ glycosyl hydrolase 18, với cấu trúc hình thùng (βα) chứa các gốc xúc tác Asp125, Glu127 và Tyr182. Nghiên cứu cũng khám phá các đặc điểm sinh hóa và cấu trúc của PPL2, bao gồm việc nó là một protein đơn phân, không glycosyl hóa, với khối lượng phân tử khoảng 29.4 kDa.

Mục lục chi tiết:

• CDNA cloning and 1.75 Å crystal structure determination of PPL2, an endochitinase and N-acetylglucosamine-binding hemagglutinin from Parkia platycephala seeds
• Keywords
• Correspondence
• Parkia platycephala lectin 2 was purified from Parkia platycephala (Leguminosae, Mimosoideae) seeds by affinity chromatography and RP-HPLC.
• Equilibrium sedimentation and MS showed that Parkia platycephala lectin 2 is a nonglycosylated monomeric protein of molecular mass 29 407 ± 15 Da, which contains six cysteine residues engaged in the formation of three intramolecular disulfide bonds.
• Parkia platycephala lectin 2 agglutinated rabbit erythrocytes, and this activity was specifically inhibited by N-acetylglucosamine.
• In addition, Parkia platycephala lectin 2 hydrolyzed β(1-4) glycosidic bonds linking 2-acetoamido-2-deoxy-β-D-glucopyranose units in chitin.
• The full-length amino acid sequence of Parkia platycephala lectin 2, determined by N-terminal sequencing and cDNA cloning, and its three-dimensional structure, established by X-ray crystallography at 1.75 Å resolution, showed that Parkia platycephala lectin 2 is homologous to endochitinases of the glycosyl hydrolase family 18, which share the (βα) barrel topology harboring the catalytic residues Asp125, Glu127, and Tyr182.
• Abbreviations
• CTAB, cetyl triethylammonium bromide; GlcNac, N-acetyl-D-glucosamine; GSP, gene-specific forward primer; HPAEC-PAD, high-pH anion exchange chromatography with pulsed amperometric detection; PE, pyridylethylated; PPL1, Parkia platycephala lectin 1; PPL2, Parkia platycephala lectin 2; PTC, phenylisothiocyanate; PTH, phenylthiohydantoin.
• Lectins comprise a heterogeneous class of (glyco)proteins that possess one noncatalytic domain that binds carbohydrates in a specific and reversible manner without altering their covalent structure [1].
• Lectins decipher the glycocodes encoded in the structure of glycans in processes such as cell communication, host defense, fertilization, development, parasitic infection, tumor metastasis, and plant defense against herbivores and pathogens [2].
• Mechanisms for sugar recognition have evolved independently in a restricted number of protein folds (e.g. jelly roll domain, C-type lectin fold, β-propeller, β-trefoil motif, β-prism I and II domains, Ig domains, β-sandwich, mixed αβ structure, and hevein domain) [1,3] (for a complete catalog of carbohydrate-binding protein domains, please consult the 3D Lectin Database at http://www.cermav.cnrs.fr/lectines).
• In plants, most of the currently known lectins can be placed in seven families of structurally and evolutionarily related proteins [1].
• The seed lectins of leguminous plants constitute the largest and most thoroughly studied lectin family.
• These lectins have represented paradigms for establishing the structural basis [4-9] and thermodynamics [10-13] of selective sugar recognition.
• Most studies on lectins from Leguminosae involve members of the Papilionoideae subfamily, whereas investigations on lectins of the other two subfamilies, Caesalpinoideae and Mimosoideae, are scarce.
• Indeed, to date, the only lectins from the Mimosoideae that have been functionally and structurally characterized are those from seeds of species of the genus Parkia, including Parkia speciosa [14], Parkia javanica [15], Parkia discolor [16] and the glucose/mannose-specific lectin from Parkia platycephala seeds [17-21].
• Parkia (Leguminosae, Mimosoideae), regarded as the most primitive group of leguminous plants [22], is a pantropical genus of trees comprising about 30 species found in the neotropics from Honduras to south-eastern Brazil, West Africa, the northern part of Malaysia and the south of Thailand.
• Parkia platycephala is an important forage tree growing in parts of north-eastern Brazil.
• The seed lectin from Parkia platycephala is a 47.9-kDa single-chain nonglycosylated mosaic protein composed of three tandemly arranged jacalin-related β-prism domains [19,20].
• The sugar-binding specificity of Parkia platycephala lectin towards mannose, an abundant building block of surface-exposed glycoconjugates of viruses, bacteria, and fungi, suggests a role for the Parkia platycephala lectin in defense against plant pathogens [1].
• Moreover, the Parkia platycephala lectin also shows sequence similarity with stress-upregulated and pathogen-upregulated defense genes of a number of different plants, suggesting a common ancestry for jacalin-related lectins and inducible defense proteins [19].
• In addition to using lectins, whose precise role in plant defense remains to be determined [23,24, and references cited], plants defend themselves against pathogens (i.e. fungi) secreting pathogenesis-related enzymes, such as xylanases and chitinases, which degrade the pathogen’s cell wall [25-27].
• In a previous article we have reported the presence of an endochitinase in Parkia platycephala seeds [28].
• Now, we have determined its complete amino acid sequence by a combination of Edman degradation and cDNA cloning, and report its biochemical characterization and the determination of its crystal structure.
• Our results show that this protein, termed Parkia platycephala lectin 2 (PPL2), is homologous to endochitinases of the glycosyl hydrolase family 18 that exhibit rabbit erythrocyte-agglutinating, N-acetylglucosamine-binding and chitin-hydrolyzing activities.
• Results and Discusion
• PPL2, a nonglycosylated and monomeric GlcNAc-binding hemagglutinin
• PPL2 was purified from Parkia platycephala seeds by affinity chromatography on either Red-Sepharose (Fig. 1A) or chitin-Sepharose.
• The protein agglutinated trypsin-treated rabbit erythrocytes (128 hemagglutinating units mg⁻¹), and this activity was abolished by 19 mm N-acetyl-D-glucosamine (GlcNac).
• Other sugars, such as glucose, mannose, galactose, fucose and N-acetyl-D-galactosamine, displayed only partial hemagglutination inhibitory activity at much higher concentrations (> 75 mm) than GlcNac.
• Moreover, the glycoproteins bovine thyroglobulin, ovine submaxillary mucin, bovine fetuin and bovine asialofetuin were devoid of hemagglutination inhibitory activity.
• Bovine thyroglobulin contains nine complex glycosylation sites and four high-mannose oligosaccharides [29].
• Ovine submaxillary mucin is a glycoprotein bearing a high density of O-linked oligosaccharides expressing sialyl Tn antigens and sialyl core 3 sequences [30].
• Bovine fetuin contains three N-linked glycosylation sites occupied with trisialylated, tetrasialylated or pentasialylated trianntennary structures, and three monosialylated or disialylated O-linked saccharides [31-33].
• We thus concluded that PPL2 represented an N-acetylglucosamine-binding hemagglutinin.
• The apparent molecular masses of both native and reduced PPL2 determined by SDS/PAGE were 30 kDa (Fig. 1A, insert).
• The molecular mass of native PPL2, measured by MALDI-TOF MS, was 29 407 ± 15 Da (Fig. 1A).
• This value was not altered upon incubation of the denatured, but nonreduced, protein with the alkylating reagent 4-vinylpyridine.
• On the other hand, the same treatment after reduction of the protein with dithiothreitol changed the molecular mass of PPL2 to 30 052 ± 15 Da (Fig. 1B).
• The mass increment of about 645 Da indicated that PPL2 had incorporated six pyridylethyl groups.
• The combined data clearly showed that PPL2 contained six cysteine residues engaged in the formation of three intramolecular disulfide bonds.
• Amino acid compositional analysis of the purified protein (Table 1) was in agreement with this conclusion.
• The estimated apparent molecular mass for PPL2 on a calibrated size-exclusion chromatographic column was 12 kDa, indicating that the protein had an anomalous elution profile.
• Molecular mass determinations by size-exclusion chromatography are dependent on the hydrodynamic properties of the molecule, and, in addition, interaction of the protein with the matrix may also introduce large errors into the estimated molecular mass.
• Thus, we carried out a more rigorous analysis of the aggregation state of PPL2 employing analytic ultracentrifugation equilibrium sedimentation, a technique that is firmly based in thermodynamics and does not therefore rely on calibration or on making assumptions concerning the shape of the protein.
• Using this approach, the apparent molecular mass of the PPL2 lectin in solutions with pH in the range 2.5-8.5 was 34 ± 3 kDa (Fig. 1B, insert).
• This figure, in conjunction with the MS analyses, showed that the protein behaved as a pH-independent monomeric protein.
• Carbohydrate analysis performed by GLC (data not shown) failed to show the presence of any amino or neutral monosaccharide, strongly indicating that PPL2 was a nonglycosylated protein.
• PPL2 displays chitinase activity
• Edman degradation analysis of reduced and pyridylethylated protein yielded the first 42 amino acid residues of PPL2: GGIVVYWGQNGGEGTLTSTCESGLYQIVNIAFLSQFGGGRRP.
• A BLAST analysis (http://www.ncbi.nlm.nih.gov/blast/) revealed extensive (up to approximately 75%) similarity with a large number of plant chitinase sequences deposited in the publicly accessible protein databases, such as the basic chitinase III from Nicotiana tabacum (P29061), an acidic chitinase from Glycine max (BAA77677), chitinase b from Phytolacca americana (Q9S9F7), chitinase from P.so.phocarpus tetragonolobus (BAA08708), chitinases from Vitis vinifera (CAC14014), basic chitinase from Vigna unguiculata (Q43684), and chitinase B from leaves of pokeweed (Q9S9F7).
• All of these proteins are poly [1,4-(N-acetyl-β-D-glucosaminide)] glycanhydrolases of the glycosyl hydrolase family 18 (EC 3.2.1.14) [34] (http://www.sanger.ac.uk/cgi-bin/Pfam/getacc?PF00704), whose prototype is hevamine, isolated from the rubber tree [35,36].
• The possible chitinase activity of PPL2 was investigated by quantitative GC determination of the amount of GlcNac released using chitin as substrate.
• PPL2 released 3 µg of GlcNac·h⁻¹·(mg protein)⁻¹.
• In comparison, commercial Streptomyces griseus chitinase exhibited an activity of 80 µg of GlcNac·h⁻¹·(mg protein)⁻¹, and the GlcNac-specific agglutinins from wheat germ (WGA) and Urtica dioica (UDA) did not show any chitinase activity.
• Peracetylated GlcNac (retention time 33.60 min) was observed in the reaction mixtures containing PPL2 or Streptomyces griseus chitinase but not in those reaction mixtures to which WGA or UDA were added.
• These results demonstrated that PPL2 was indeed an active chitinase able to hydrolyze the β(1-4) glycosidic bond linking the GlcNac units of chitin.
• In order to determine whether PPL2 presented chitinase activity only for the nonreducing end of chitin (exochitinase activity) or also had the ability to hydrolyze internal β(1-4) glycosidic linkages (an endochitinase activity), 40 µL of the reaction mixture used for the chitinase assay were analyzed by Dionex high-pH anion exchange chromatography using a CarboPac PA-100 column.
• The elution times of three major analytes present in the reaction mixture (3.93, 4.84 and 5.58 min) matched those of the standard carbohydrates GlcNac, (GlcNac)2 and (GlcNac)3 (3.86, 4.84 and 5.58 min, respectively).
• This result demonstrated an endochitinase activity for PPL2.
• The exact mechanism of glycoside hydrolysis (e.g. with retention or not of the β-anomeric configuration of the products) remains to be established, however.
• The finding that PPL2 exhibited GlcNac-dependent hemagglutination and endochitinase activities was striking but not without precedent.
• The acidic chitinase BjCHI1 from Brassica juncea showed hemagglutination ability [37].
• However, BjCHI1 is a unique chitinase with two chitin-binding domains, and both chitin-binding domains are essential for agglutination [38].
• On the other hand, PPL2 is a single-domain protein.
• Hence, PPL2 may possess at least two carbohydrate-binding sites.
• One of them probably corresponds to the catalytic site, whereas the other one(s) remain to be characterized.
• Plant chitinases constitute a class of pathogenesis-related proteins that play an important role in defense against pathogens through degradation of chitin present in the fungal cell wall and in insect cuticles [37,39].
• The first characterization of a chitinase in the Mimosoideae subtribe, an antifungal chitinase from Leucaena leucocephala has been reported only recently [40].
• This protein belongs to the class I chitinases of the glycosyl hydrolase family 19, and is, thus, structurally unrelated to PPL2.
• It is noteworthy that the seeds of Parkia platycephala contain two different lectins: the mannose/glucose-specific PPL1 [19,21] and the GlcNac-binding lectin with chitinase activity, PPL2, described here.
• The fact that mannose is an abundant building block of surface-exposed glycoconjugates of viruses, bacteria and fungi supports the view that PPL, and other mannose-recognizing lectins, play a role in plant defense against pathogens [1].
• Specifically, the planar array of carbohydrate-binding sites on the rim of the toroid-shaped structure of the Parkia platycephala lectin dimer [21] immediately suggested a mechanism to promote multivalent interactions leading to cross-linking of carbohydrate ligands as part of the host strategy against phytopredators and pathogens.
• The presence of two unrelated lectins in plant seeds has been also reported in Canavalia ensiformis (Leguminosae): concanavalin A, a prototypic glucose/mannose-specific legume lectin built by the jellyroll fold [1,7], and concanavalin B, which, although it shares about 40% sequence identity with plant chitinases belonging to glycosyl hydrolase family 18, has not been shown to have any chitinase activity [41].