Edmund W. Czerwinski

Identification of the Structural and Functional Domains of MutY, an Escherichia coli DNA Mismatch Repair Enzyme

The linear amino acid sequences of the Escherichia coli DNA repair proteins, MutY and endonuclease III, show significant homology, even though these enzymes recognize entirely different substrates. In this study, proteolysis and molecular modeling of MutY were used to elucidate its domain organization. Proteolysis by trypsin cleaved the enzyme into 26- and 13-kDa fragments. NH2-terminal sequencing showed that the p13 domain begins at Gln226, indicating that the COOH-terminal portion of MutY, absent in endonuclease III, is organized as a separate domain. The large p26 domain is almost equivalent to the size of endonuclease III. Binding activity of the p26 domain to a DNA substrate containing an A·G mismatch was comparable with that of the intact enzyme. In vitro studies show that the p26 domain retains adenine glycosylase and AP lyase activity on DNA containing undamaged adenine opposite guanine or 8-oxo-7,8-dihydro-2′-deoxyguanine. Although the activity was somewhat reduced, the above results show that the critical amino acid residues involved in substrate binding and catalysis are present in this domain. The structure predicted by molecular modeling indicates that the region of MutY (Met1-Trp216), which is homologous to endonuclease III exhibits a two domain structure, even though this portion is resistant to proteolysis by trypsin.

Calcium phosphate crystal formation in Escherichia coli from human urine: An in vitro study

Escherichia coli with no demonstrable urease activity was inoculated into filter sterilized urine obtained from a healthy volunteer subject with no history of stone disease and then incubated at 37 degrees C. Bacteria were recovered at intervals between 1 and 10 days. Urinary pH was stable as compared to control urines and spontaneous crystal precipitation was not noted in controls. Recovered organisms were analyzed by x-ray powder diffractometry. An uncharacterized mineral phase (UMP) was first evident after 6 days. Calcium phosphate in the form of brushite and hydroxyapatite was apparent at 7 and 10 days respectively. This suggests a role for bacteria in calcium phosphate crystal formation in urine apart from urease activity and may contribute to the calcium phosphate component of urinary calculi.

Plant-Expressed Recombinant Mountain Cedar Allergen Jun a 1 Is Allergenic and Has Limited Pectate Lyase Activity

Background: Mountain cedar (Juniperus ashei) pollen commonly causes a winter time allergic rhinitis in the central USA. Jun a 1 is the dominant allergenic protein, but biologically active recombinant Jun a 1 has not been successfully expressed, despite numerous attempts with several expression systems. Method: Jun a 1 cDNA was inserted into a tobacco mosaic virus vector and transferred to Agrobacterium tumefaciens. Bacteria were syringe-inoculated into leaves of Nicotiana benthamiana (agroinoculation). The interstitial (apoplastic) fluid containing Jun a 1 was isolated. The recombinant protein was analyzed by SDS-PAGE, N-terminal sequencing and MALDI-TOF to confirm identity. Immunogenicity was examined with IgE from allergic patient’s sera, mouse monoclonal anti-Jun a 1 antibodies, IgE-binding inhibition and by degranulation of RBL SX-38 cells sensitized with sera from allergic patients. Pectate lyase activity was assayed by capillary zone electrophoresis and mass spectrometry analysis. Results: Recombinant Jun a 1 was recovered in good quantity (100 µg/g leaf material), was confirmed as Jun a 1, bound IgE from sera from cedar hypersensitive patients and inhibited IgE binding to native Jun a 1. Jun a 1 mutants were created and their pectate lyase activity quantified. For the first time, Jun a 1 pectate lyase activity was demonstrated, which may explain the necrosis seen on host plants, which was similar to that of control plants expressing banana pectate lyase. Conclusions: A means of producing recombinant Jun a 1 is now available for structure/function studies and potentially for diagnostic and therapeutic uses.

Crystallization and preliminary X-ray diffraction analysis of Jun a 1, the major allergen isolated from pollen of the mountain cedar Juniperus ashei

Jun a 1, the major allergen of pollen from the mountain cedar Juniperus ashei, has been crystallized using the hanging-drop vapor-diffusion method at 277 K. The crystals are monoclinic, space group P2(1), with unit-cell parameters a = 53.38, b = 113.48, c = 72.44 A, beta = 96.36 degrees and four molecules in the unit cell. A complete 2.5 A data set has been collected at 100 K with X-rays from a Cu Kalpha rotating-anode generator.