Stability Effects Associated With the Introduction of a Partial and a Complete Ca2+-binding Site Into Human Lysozyme

Two mutants of human lysozyme were synthesized. Mutant A92D, in which Ala92 was substituted by Asp, contains a partial Ca2+-binding site and mutant M4, in which Ala83, Gln86, Asn88 and Ala92 were replaced by Lys, Asp, Asp and Asp respectively, contains the complete Ca2+-binding site of bovine alpha-lactalbumin. The Ca2+-binding constants of wild type human lysozyme and of mutants A92D and M4, measured at 25-degrees-C and pH 7.5, were 2(+/- 1) x 10(2) M-1, 8(+/- 2) x 10(3) M-1 and 9(+/- 0.5) X 10(6) M-1 respectively. Information gathered from microcalorimetric and CD spectroscopic measurements indicates that the conformational changes of the M4 mutant lysozyme, induced by Ca2+ binding, are smaller than those observed for bovine alpha-lactalbumin and for the Ca2+-binding equine lysozyme. At pH 4.5, the thermostability of both the apo and Ca2+ forms of the A92D human was decreased in comparison with that of native human lysozyme. In particular, within the apo form of this mutant an alpha-helix-containing sequence was destabilized. In contrast, at the same pH the thermostability of the apo and Ca2+ forms of the M4 mutant lysozyme was increased. The epsilon-ammonium group of the Lys83 side chain is assumed to be responsible for the stabilization of the apo form of this mutant

Lmp2(+) Proteasomes Are Required for the Presentation of Specific Antigens To Cytotoxic T-lymphocytes

Background: Major histocompatibility complex (MHC) class I molecules present short peptides generated by intracellular protein degradation to cytotoxic T lymphocytes (CTL). The multisubunit, non-lysosomal proteinases known as proteasomes have been implicated in the generation of these peptides. Two interferon-gamma (IFN-gamma)-inducible proteasome subunits, LMP2 and LMP7, are encoded within the MHC gene cluster in a region associated with antigen presentation. The incorporation of these LMP subunits into proteasomes may alter their activity so as to favour the generation of peptides able to bind to MHC class I molecules. It has been difficult, however, to demonstrate a specific requirement for LMP2 or LMP7 in the presentation of peptide epitopes to CTL. Results: We describe a T-cell lymphoma, termed SP3, that displays a novel selective defect in MHC class I-restricted presentation of influenza virus antigens. Of the MHC-encoded genes implicated in the class I pathway, only LMP2 is underexpressed in SP3 cells. Expression of IFN-gamma in transfected SP3 cells simultaneously restores LMP2 expression and antigen presentation to CTL. Expression of antisense-LMP2 mRNA in these IFN-gamma-transfected cells selectively represses antigen recognition and the induction of surface class I MHC expression. Moreover, the expression of this antisense-LMP2 mRNA in L929 fibroblast cells, which constitutively express LMP2 and have no presentation defect, blocks the presentation of the same influenza virus antigens that SP3 cells are defective in presenting. Conclusions: Our results show that the LMP2 proteasome subunit can directly influence both MHC class I-restricted antigen presentation and class I surface expression

A New Production Method for Glucose-oxidase

The recombinant yeast expression plasmid, pSGO2, has been constructed containing a hybrid yeast alcohol dehydrogenase II-glyceraldehyde-3-phosphate dehydrogenase promoter, the glucose oxidase presequence and the mature glucose oxidase coding sequence. When transformed into yeast, this plasmid directs the synthesis and secretion of more than 1.5 g l-1 of active glucose oxidase. Analysis of the yeast-derived enzyme shows extensive N-linked glycosylation in comparison to the A. niger protein. Both proteins show similar specific activities in terms of U mg-1 of protein