Random mutagenesis and screening for enzymatic activity has been used to engineer horse heart myoglobin to enhance its intrinsic peroxidase activity. A chemically synthesized gene encoding horse heart myoglobin was subjected to successive cycles of PCR random mutagenesis. The mutated myoglobin gene was expressed in Escherichia coli LE392, and the variants were screened for peroxidase activity with a plate assay.

SummaryA maternal plant exquisitely promotes the success of its offspring by orchestrating embryo development and endowing protection even after the embryos mature. It uses ovule integuments for physical and physiological contact with the developing embryo and for subsequently equipping the seed with a seed coat (testa). The testa is developmentally and metabolically dynamic, but its molecular biology is not well understood. We show here that the inner integument in Brassica napus undergoes organized development and then programmed cell death (PCD), as evident from vacuolation, starch mobilization, DNA fragmentation and eventual compression. We have identi®ed a cysteine proteinase gene (BnCysP1) that is expressed only in the inner integument as it undergoes PCD, well before the embryo begins storage protein synthesis. Two paralogous Cys proteinases have been recruited in rapeseed for the PCD of testa and for leaf senescence, and these differ 25% in their primary structure and post-translational modi®cations. Despite Arabidopsis being closely related to rapeseed, and an indication of developmental compression of its inner integument, the Arabidopsis genome is suggestive of only one Cys proteinase that shows »72% identity to BnCysP1. It is, however, leaf senescence-associated, and the other Cys proteinases are <52% identical. BnCysP1 also differs from ricinosome-deployed PCD Cys endopeptidases in lacking the hallmark KDEL tail and being glycosylated. BnCysP1, one of the very few plant genes known to function only in the seed coat, will be useful in dissecting post-fertilization development of this important organ in rapeseed.

Cruciferin (a 12 S globulin) is the most abundant storage protein in the seeds of Arabidopsis thaliana (thale cress) and other crucifers, sharing structural similarity with the cupin superfamily of proteins. Cruciferin is synthesized as a precursor in the rough endoplasmic reticulum. Subunit assembly is accompanied by structural rearrangements involving proteolysis and disulfidebond formation prior to deposition in protein storage vacuoles. The A. thaliana cv. Columbia genome contains four cruciferin loci, two of which, on the basis of cDNA analysis, give rise to three alternatively spliced variants. Using MS, we confirmed the presence of four variants encoded by genes At4g28520.1, At5g44120.3, At1g03880.1 and At1g3890.1 in A. thaliana seeds. Two-dimensional gel electrophoresis, along with immunological detection using anti-cruciferin antiserum and antibodies against phosphorylated amino acid residues, revealed that cruciferin was the major phosphorylated protein in Arabidopsis seeds and that polymorphism far exceeded that predicted on the basis of known isoforms. The latter may be attributed, at least in part, to phosphorylation site heterogeneity. A total of 20 phosphorylation sites, comprising nine serine, eight threonine and three tyrosine residues, were identified by MS. Most of these are located on the IE (interchain disulfide-containing) face of the globulin trimer, which is involved in hexamer formation. The implications of these findings for cruciferin processing, assembly and mobilization are discussed. In addition, the protein phosphatase 2C-impaired mutant, abi1-1, was found to exhibit increased levels of cruciferin phosphorylation, suggesting either that cruciferin may be an in vivo target for this enzyme or that abi1-1 regulates the protein kinase/phosphatase system required for cruciferin phosphorylation.

Summary. Background: The plasmin(ogen) and complement systems are simultaneously activated at sites of tissue injury, participating in hemostasis, wound healing, inflammation and immune surveillance. In particular, the C3 proteolytic fragment, iC3b, and its degradation product C3dg, which is generated by cleavage by factor I (FI) and the cofactor complement receptor CR1, are important in bridging innate and adaptive immunity. Via a thioester (TE) bond, iC3b and C3dg covalently tag pathogens, modulating phagocytosis and adaptive immune responses. Objective: To examine plasmin-mediated proteolysis of iC3b, and to evaluate the functional consequences, comparing the effects with products generated by FI/CR1 cleavage of iC3b. Methods: Dose-dependent and time-dependent plasmin-mediated cleavage of iC3b were characterized by analytical gel electrophoresis. The properties of the resultant TE bond-containing fragments on phagocytosis and induction of pro-inflammatory cytokines were measured in cell culture systems. Results: At low concentrations, plasmin effectively cleaves iC3b, but at numerous previously undescribed sites, giving rise to novel C3c-like and C3dg-like moieties, the latter of which retain the TE bond. When attached to zymosan or erythrocytes and exposed to THP-1 macrophages, the C3dg-like proteins behave almost identically to the bona fide C3dg, yielding less phagocytosis as compared with the opsonin iC3b, and more macrophage secretion of the pro-inflammatory cytokine, IL-12. Conclusion: Plasmin cleavage of iC3b provides a complement regulatory pathway that is as efficient as FI/CR1 but does not require a cellular cofactor.