Cloning and Characterization of Physarum polycephalumTectonins

Abstract

Previous investigators have reported the presence of two dominant proteins, tectonin I (25 kDa) and tectonin II (39 kDa), in nuclei and nuclear matrix from plasmodia of Physarum polycephalum. We demonstrate, by a modification of the nuclear isolation protocol and by protease sensitivity, that the tectonins are not nuclear proteins but rather are located on the exterior surface of the plasma membrane.We report the sequences of cDNAs of tectonins I and II, which encode 217 and 353 amino acids, respectively. Tectonin I is homologous to the C-terminal two-thirds of tectonin II. Both proteins contain six tandem repeats that are each 33-37 amino acids in length and define a new consensus sequence. Homologous repeats are found in L-6, a bacterial lipopolysaccharide-binding lectin from horseshoe crab hemocytes. The repetitive sequences of the tectonins and L-6 are reminiscent of the WD repeats of the ␤-subunit of G proteins, suggesting that they form ␤-propeller domains. Tectonin II has an additional N-terminal domain that includes a 47-residue sequence highly similar to the galactoside-binding sequence of the B-chain of ricin. The tectonins may be lectins that function as part of a transmembrane signaling complex during phagocytosis.In its plasmodial form, the myxomycete Physarum polycephalum exists as a multinucleated syncytium that feeds on bacteria and organic detritus by phagocytosis. The many nuclei within a single plasmodium progress through the cell cycle synchronously, and at the end of the G 2 phase undergo closed mitosis. Because of these characteristics, several investigators have examined the P. polycephalum nuclear matrix (1-4) and reported that, as with mammalian nuclear matrix, the P. polycephalum matrix contained a number of proteins ranging from approximately 40 kDa to more than 100 kDa but that it differed from mammalian nuclear matrix by having two dominant proteins with reported molecular masses of 23-28 and 35-38 kDa as determined by SDS-PAGE.1 The same proteins have also been found associated with purified rDNA chromatin (5). We have termed these proteins tectonins I and II, respectively.In the present study we report the cloning and sequencing of the cDNAs for the tectonins, and we use trypsin digestion of cell fractions to assess their localization in the plasmodium. We find that the tectonins are not nuclear proteins but instead are located on the plasmodial surface, and we report a method for purifying P. polycephalum nuclei not contaminated with the tectonins.The tectonins share with lectin L-6 of horseshoe crab hemocytes (6) six repeats of a novel consensus sequence that may form a ␤-propeller structure. Additionally, tectonin II contains in its N-terminal region a sequence similar to the galactosebinding domain of the B-chain of the plant toxin ricin (7). The tectonins may share with lectin L-6 the ability to recognize the outer membrane lipopolysaccharide of Gram-negative bacteria for phagocytosis and utilize an additional affinity for galactose to expand the number of ligands that they re...

A Sperm-Associated WD Repeat Protein Orthologous to Chlamydomonas PF20 Associates with Spag6, the Mammalian Orthologue of Chlamydomonas PF16

“…WD domains are made up of highly conserved repeating units usually ending with Trp-Asp (WD). They are found in all eu- (12,21,22,30,38). An increasing number of axoneme proteins have been found to contain WD repeats, and evidence suggests that this feature is important for regulation of flagellar motility (22).…”

Emergence and Disappearance of an Immune Molecule, an Antimicrobial Lectin, in Basal Metazoa

“…Like the crab molecules, sponge LEC_SUBDO comprises a predicted transmembrane region, suggesting that the lectin is cell surfaceassociated. In addition, six distinct hydrophilic regions are present in the sponge molecule, corresponding to six tandem repeats in the tachylectins; these are absent in tectonins, related molecules found in the Mycetozoa P. polycephalum (39). To rule out the possibility that the cloned LEC_SUBDO is not of sponge origin and might have been introduced into the cDNA library from commensal animals, different S. domuncula specimens were analyzed by reverse transcription-PCR.…”

“…tonins are present in the nucleus (39). No (potential) function is known for the vertebrate molecules (38).…”

Methylated glycans as conserved targets of animal and fungal innate defense

“…The Tectonin domain comprises a 33-37 amino acid consensus sequence that was first reported in Tectonin I and II proteins from the slime mold Physarum polycephalum (5). To date, Tectonins from various organisms including sponge, horseshoe crab, fish, and human have been characterized (6)(7)(8)(9)(10)(11)(12).…”