Defining the repeating elements in the cysteine-rich region (CRR) of the CD18 integrin beta 2 subunit.

The cysteine-rich region (CRR) of the integrin beta subunits is organised into four repeating elements. By expression of a panel of truncated beta 2 subunits, and CRR segments fused to the C-terminal end of a CD4 soluble fragment, the segment required for the expression of two monoclonal antibody conformational epitopes was determined. This segment, E482-Q574, contains 16 cysteines representing two repeating units. We have thus defined the CRR unit motif of 'xC---C---C---CxCxxCxC---Cx', where 'x' represents a single residue, and '---' represents a stretch of four to 14 residues.

The effects of cysteine to alanine mutations of CD18 on the expression and adhesion of the CD11/CD18 integrins.

Of the 56 cysteines in the extracellular domain of the CD18 antigen (beta2 integrin subunit), corresponding ones are not found in 12 positions in the beta4, beta7, or beta8 integrin subunits. These 12 cysteines were mutated to alanines, either singly or in pairs, in CD18. All these mutants can support the expression of all three CD11/CD18 integrins. Transfectants expressing these variant integrins are generally more adhesive than the wild-type, suggesting that the cysteine residues, perhaps by engaging in disulphide bonds, may contribute to the maintenance of the CD11/CD18 integrins in a resting state.

A novel point mutation in CD18 causing the expression of dysfunctional CD11/CD18 leucocyte integrins in a patient with leucocyte adhesion deficiency (LAD).

Leucocyte adhesion deficiency type 1 (LAD-1) is characterized by the incapacity of leucocytes to carry out their adhesion functions via their CD11/CD18 antigens, which are also referred to as the leucocyte integrins. The patients generally suffer from poor wound healing and recurrent bacterial and fungal infections. In severe cases, the infections are often systemic and life-threatening. A LAD patient (AW) of moderate phenotype has been identified but, unlike most other cases, the level of CD11/CD18 antigens on her leucocytes are uncharacteristically high for a LAD patient. Molecular analysis revealed that she is a compound heterozygote for CD18 mutations. She has inherited a D231H mutation from her father and a G284S mutation from her mother. By transfection studies, it was established that the G284S mutation does not support CD11/CD18 antigen expression on the cell surface. In contrast, the D231H mutation does not affect CD18 forming integrin heterodimers with the CD11 antigens on the cell surface. However, the expressed integrins with the D231H mutation are not adhesive to ligands.

The effects of targeting the vaccinia virus B5R protein to the endoplasmic reticulum on virus morphogenesis and dissemination.

The consequence of redirecting the vaccinia virus (VV) B5R protein to the endoplasmic reticulum (ER) has been investigated by the addition of an ER retrieval signal KKSL (K(2)X(2)) to the B5R C-terminus. This mutant B5R gene and a version of the gene with the inactive ER retrieval sequence KKSLAL (K(2)X(4)) were inserted into the thymidine kinase locus of a VV mutant lacking the B5R gene, vDeltaB5R. Similar levels of B5R protein were made by each virus, but the B5R-K(2)X(2) protein remained sensitive to endoglycosidase H and colocalised with protein disulphide isomerase in the ER. In contrast, the B5R-K(2)X(4) protein colocalised with 1, 4-galactosyltransferase in the trans-Golgi network. Electron microscopy revealed that even when the B5R protein was redirected to the ER, intracellular mature virus particles were wrapped by cellular membranes to form intracellular enveloped virus particles, although more incompletely wrapped particles were evident compared with wild type. These intracellular enveloped virus particles were, however, unable to efficiently induce the polymerisation of actin and the plaque size formed by vB5R-K(2)X(2) was small. Nevertheless, the amount and specific infectivity of EEV produced by vB5R-K(2)X(2) were similar to those of wild type, despite the dramatic reduction in the amount of B5R protein present in vB5R-K(2)X(2) EEV.