No abstract

Ubiquitination regulates the stability and/or activity of numerous cellular proteins. The corollary is that de-ubiquitinating enzymes, which 'trim' polyubiquitin chains from specific substrate proteins, play key roles in controlling fundamental cellular activities. Ubiquitin is essential at several stages during the activation of NF-kappaB (nuclear factor kappaB), a central co-ordinator of inflammation and other immune processes. Ubiquitination is known to cause degradation of the inhibitory molecule IkappaBalpha (inhibitor of kappaB). In addition, activation of TRAF (tumour-necrosis-factor-receptor-associated factor) and IKKgamma (IkappaB kinase gamma)/NEMO (NF-kappaB essential modifier) signal adaptors relies on their modification with 'nonclassical' forms of polyubiquitin chains. Ubiquitin also plays a key role in determining cell fate by modulating the stability of numerous pro-apoptotic or anti-apoptotic proteins. The zinc-finger protein A20 has dual functions in inhibiting NF-kappaB activation and suppressing apoptosis. The molecular mechanisms of these anti-inflammatory and cytoprotective effects are unknown. Here we demonstrate that A20 is a de-ubiquitinating enzyme. It contains an N-terminal catalytic domain that belongs to the ovarian-tumour superfamily of cysteine proteases. A20 cleaved ubiquitin monomers from branched polyubiquitin chains linked through Lys48 or Lys63 and bound covalently to a thiol-group-reactive, ubiquitin-derived probe. Mutation of a conserved cysteine residue in the catalytic site (Cys103) abolished these activities. A20 did not have a global effect on ubiquitinated cellular proteins, which indicates that its activity is target-specific. The biological significance of the catalytic domain is unknown.

Samples of breast milk and serum were taken from 29 women at various stages of lactation before and after they had ingested 1 raw egg and half a pint of cow’s milk. The samples were analysed for cow’s milk and egg proteins using a solid-phase radioimmunoassay. β-Lactoglobulin, ovalbumin (OA) and ovomucoid were detected in breast milk from 10 out of 19, 13 out of 22 and 7 out of 9 women, respectively, in concentrations ranging from 110 pg/ml to 6.4 ng/ml. Maximum levels in breast milk were attained 4 or 6 h after ingestion and in serum 1–2 h earlier. The OA in breast milk was of normal molecular size (43,500 daltons) and indistinguishable from native OA in the radioimmunoassay. Gel filtration of whole breast milk or high molecular weight fractions at pH 2.6 failed to reveal additional OA, suggesting that immune complexes were not present. In serum, OA was detected both in its native form and in immune complexes. The possible significance of these antigens in the suckling infant is discussed.

The integrin alpha M290 beta 7 on the surface of a T cell hybridoma, MTC-1, mediated adhesion of these cells to the mouse epithelial cell line CMT93. This interaction was critically dependent on the presence of divalent cations; Mn2+ strongly promoted adhesion, Ca2+ was ineffective and Mg2+ gave intermediate results. Antibodies to molecules on the surface of CMT93 cells were tested for inhibition of adhesion. One monoclonal antibody (mAb) against E-cadherin, ECCD-2, was found to have significant inhibitory activity. Other mAb to E-cadherin and antibodies to other molecules had no effect. To show that inhibition by ECCD-2 was specific for adhesion mediated by alpha M290 beta 7, MTC-1 cells were induced to adhere to CMT93 via the LFA-1/ICAM-1 pathway. For this purpose, the epithelial cells were treated with interferon-gamma and tumor necrosis factor-alpha to induce ICAM-1 expression and, in addition, alpha M290 beta 7 on MTC-1 cells was down-regulated by culturing the cells in the absence of transforming growth factor beta. Under these circumstances adhesion of MTC-1 cells to CMT93 was inhibited by an antibody to LFA-1 but not by ECCD-2. Transfection of mouse L cells with cDNA for mouse E-cadherin enabled MTC-1 cells to adhere to them through the alpha M290 beta 7 integrin; this interaction was inhibited both by ECCD-2 and by blocking antibody against the integrin. These data strongly suggest that E-cadherin is a principal ligand for alpha M290 beta 7.

A new surface molecule has been discovered on mouse intestinal intraepithelial lymphocytes (IEL) using a rat anti-mouse IEL monoclonal antibody, M290. It was expressed at high levels on nearly all IEL and on a majority of T cells in the gut lamina propria. M290 stained, with lower intensity, a small minority of T cells in other lymphoid tissues. Expression was biased towards the CD8+ subset. Stimulation of peripheral T cells with mitogens did not induce expression of the new antigen but addition of transforming growth factor beta to stimulated T cells had a marked inductive effect. Sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis of IEL surface components precipitated with M290 showed principal bands at 135, 120, 28 and 24 kDa (reduced) and 135, 100, 24 and 21 kDa (nonreduced). Precipitation with antibodies to integrin subunits showed that the new molecular complex was not a member of the beta 1, beta 2, or beta 3 integrin families although all of these were represented on IEL. A 13-amino acid N-terminal sequence obtained from the 120-kDa beta subunit of the antigen prepared from an M290+ T hybridoma (MTC-1) did not show homology with integrins. Pulse-chase studies using MTC-1 cells showed that the 135-kDa alpha subunit was derived from a 147-kDa precursor. The function of this new molecular complex is not yet known.