Crystallization and preliminary X-ray study of endopolygalacturonase from the pathogenic fungus Stereum purpureum.

Crystals of endopolygalacturonase I from Stereum purpureum have been obtained by the vapour-diffusion method. Prior to crystallization work, endopolygalacturonase I was deglycosylated with endo-beta-N-acetylglucosaminidase H. The crystal diffracts to ultrahigh (0.96 A) resolution using synchrotron radiation and belongs to space group P1, with unit-cell parameters a = 37.26, b = 46.34, c = 52.05 A, alpha = 67.17, beta = 72.44, gamma = 68.90 degrees.

Determination of glycosylation sites, disulfide bridges, and the C-terminus of Stereum purpureum mature endopolygalacturonase I by electrospray ionization mass spectrometry.

Stereum purpureum endopolygalacturonase (endoPG; EC 3.2.1.15) is a causal protein for silver-leaf disease in apple trees. Endopolygalacturonase I, is a mixture of three components (Ia, Ib, and Ic) that produce three bands on SDS/PAGE but have the same polypeptide and sugar chains. Electrospray ionization mass spectrometry (ESI-MS) analysis of three endoPG I proteins and deglycosylated endoPG Ia revealed a molecular mass of 37 068, 38 285, and 39 503 for Ia, Ib, and Ic, respectively; the number of N-binding sugar chains matches that of a high-mannose type of sugar chain. Two, three, and four sugar chains are present in endoPG Ia, Ib, and Ic, respectively. Deletion of 44 amino acids from the deduced sequence occurred in the C-terminal region. Positions of the glycosylation sites and disulfide bridges were decided by tryptic digestion followed by liquid chromatography-electrospray mass spectrometry (LC-ESI-MS) analysis of reductive and nonreductive pyridylethylated endoPG I proteins. The glycosylated asparagines were determined to be Asn92 and 161; Asn92, 161, 279, or 302; and Asn92, 161, 279, and 302 in Ia, Ib, and Ic, respectively. Three disulfide bridges were noted at Cys3-Cys17, Cys175-Cys191, and Cys300-Cys303. These results are the first findings for fungal endoPG and may contribute to clarification of the relationship between stereostructure and catalytic activity.

Isolation, characterization, and sugar chain structure of endoPG Ia, Ib and Ic from Stereum purpureum.

Three endopolygalacturonases (endoPG Ia, Ib, and Ic) were isolated from the culture filtrate of Stereum purpureum, the causative fungus of apple silver-leaf disease. Their properties, including specific activities, optimum pHs, thermal stabilities, and kinetic parameters (K(m) and Vmax) were compared. Their properties were very similar to one another except for the substrate specificity and relative molecular mass. The sugar chains of endoPG Is were released by hydrazinolysis, and one major sugar chain common to endoPG Is was isolated. The pyridylamino sugar was characterized by a two-dimensional mapping method using HPLC, and identified as a high mannose type N-linked sugar chain, Man alpha 1-6(Man alpha 1-3)Man alpha 1-6(Man alpha 1-3) Man beta 1-4 GlcNAc beta 1-4 GlcNAc (designated as M5.1). Observation of the course of Western blot analysis for the proteins from the culture filtrate with endoPG I antibodies showed that the fungus secreted three endoPG Is into the culture broth during the growing period.

Antifungal Cyclodepsipeptides, W493 A and B, from Fusarium sp.: Isolation and Structural Determination.

W493 A and B, which showed strong antifungal activity, were isolated from a culture broth of Fusarium sp. The structure of W493 B was determined to be that of a cyclodepsipeptide, cyclo(3S,4R-HMTA-D-allo-Thr-L-Ala-D-Ala-L-Gln-D-Tyr-L-Ile) (1) by MS and NMR data, an amino acid analysis, and synthesis of the component. HMTA represents 3-hydroxy-4-methyltetradecanoic acid. W493 A (2) had a similar structure, except that L-Ile in 1 was replaced by L-Val. The absolute configuration of each amino acid was determined by chiral HPLC, and the sequence of the components was determined by HMBC experiments. The sequence of the two alanines was determined to be a L-Ala-D-Ala by a chiral HPLC analysis of the peptide fragment containing only one Ala residue. The absolute configuration of HMTA obtained from the hydrolysis of W493 B was determined to be 3S and 4R by comparing with four isomers prepared by enantioselective synthesis via Sharpless asymmetric epoxidation.

Cloning and sequence analysis of cDNA encoding endopolygalacturonase I from Stereum purpureum.

Endopolygalacturonase (endoPG) I was obtained from Stereum purpureum by an improved easier purification procedure. It was found that EndoPG I consisted of three glycosilated proteins with the same isoelectric point and different molecular masses, 42, 45, and 48 kDa, respectively. However, the enzymatic deglycosilation product of endoPG I gave a single band at the position corresponding to 39kDa on SDS-PAGE. Furthermore, the N-terminal amino acid sequences of three endoPGs were identical one another up to 20 residues. A cDNA library was constructed and positive cDNA clones encoding endoPG I were isolated by using antibody raised against the purified endoPG I. Nucleotide sequence analysis of the cDNA disclosed a 1212-bp open reading frame that encoded 403 amino acid residues. The N-terminal amino acid sequence (residues 1-20) of endoPG I coincided with the deduced amino acid sequence starting from the 25th residue. Therefore, the sequence of the first 24 residues represented a signal peptide and the remaining sequence, consisting of 379 residues, was the mature protein with molecular mass of 39.1 kDa. The deduced sequence of endoPG I showed 30-45% similarity in comparison with those of bacterial and fungal endoPGs, and the sequence of putative active site residues reported for the endoPGs was highly conserved in the sequence of endoPG I.

A rupture of lung metastasis of hepatocellular carcinoma causing haemothorax.

A 67 year old male with non-resectable hepatocellular carcinoma (HCC) in both lobes and liver cirrhosis was treated with transcatheter arterial embolization and regional chemotherapy. He was doing well for 18 months. He was readmitted for fever, chest pain and multiple pulmonary metastases. During interleukin-2 therapy, he suddenly developed dyspnoea and palpitation, and was in shock. Left-sided haemothorax was confirmed by draining 3 L of fresh blood. In spite of intensive care, he died within 36 h. Autopsy showed that the haemothorax was caused by rupture of one of the metastases in the upper lobe of the left lung, and that the primary HCC was totally necrotic. Survey of the literature failed to find a report of fatal bleeding from a lung metastasis of HCC.