The αE(CD103)ß7 integrin interacts with oral and skin keratinocytes in an E-cadherin-independent manner*.

The integrin αE(CD103)ß7 (αEß7) is expressed by intraepithelial lymphocytes, dendritic cells and regulatory T cells. It plays an important role in the mucosal immune system by retaining lymphocytes within the epithelium and is involved in graft rejection, immunity against tumours and the generation of gut-homing effector cells. In gut and breast, the ligand for αEß7 is E-cadherin but in human oral mucosa and skin, there is evidence that lymphocytes use an alternative, unknown, ligand. In the present study, the I domain of the human αE subunit, which contains the E-cadherin-binding site, was locked in a highly active, 'open' and an inactive, 'closed' conformation by the introduction of disulphide bonds and these domains were expressed as IgG Fc fusion proteins. αE fusion proteins recognize E-cadherin, the only known ligand for αEß7. This interaction was inhibited by an antibody that blocks the αE-binding site on E-cadherin and by the omission of Mn(2+) , which is essential for integrin function in vitro. The locked 'open' conformation of αE adhered to human oral and skin keratinocytes, including the E-cadherin-negative H376 cell line, and this was not inhibited by blocking antibody against the αEß7-binding site on E-cadherin, providing further evidence for the existence of an alternative ligand for αEß7 in skin and oral mucosa. The interaction with E-cadherin and the alternative ligand was Mn(2+) dependent and mediated by the metal ion-dependent coordination site (MIDAS) of the locked 'open'αE I domain, independently of the ß7 subunit.

Zinc-finger protein A20, a regulator of inflammation and cell survival, has de-ubiquitinating activity.

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.

Heavy chain-only antibodies are spontaneously produced in light chain-deficient mice.

In healthy mammals, maturation of B cells expressing heavy (H) chain immunoglobulin (Ig) without light (L) chain is prevented by chaperone association of the H chain in the endoplasmic reticulum. Camelids are an exception, expressing homodimeric IgGs, an antibody type that to date has not been found in mice or humans. In camelids, immunization with viral epitopes generates high affinity H chain-only antibodies, which, because of their smaller size, recognize clefts and protrusions not readily distinguished by typical antibodies. Developmental processes leading to H chain antibody expression are unknown. We show that L(-/-) (kappa(-/-)lambda(-/-)-deficient) mice, in which conventional B cell development is blocked at the immature B cell stage, produce diverse H chain-only antibodies in serum. The generation of H chain-only IgG is caused by the loss of constant (C) gamma exon 1, which is accomplished by genomic alterations in C(H)1-circumventing chaperone association. These mutations can be attributed to errors in class switch recombination, which facilitate the generation of H chain-only Ig-secreting plasma cells. Surprisingly, transcripts with a similar deletion can be found in normal mice. Thus, naturally occurring H chain transcripts without C(H)1 (V(H)DJ(H)-hinge-C(H)2-C(H)3) are selected for and lead to the formation of fully functional and diverse H chain-only antibodies in L(-/-) animals.

Convergence of Igf2 expression and adhesion signalling via RhoA and p38 MAPK enhances myogenic differentiation.

Cell-cell contact is essential for appropriate co-ordination of development and it initiates significant signalling events. During myogenesis, committed myoblasts migrate to sites of muscle formation, align and form adhesive contacts that instigate cell-cycle exit and terminal differentiation into multinucleated myotubes; thus myogenesis is an excellent paradigm for the investigation of signals derived from cell-cell contact. PI3-K and p38 MAPK are both essential for successful myogenesis. Pro-myogenic growth factors such as IGF-II activate PI3-K via receptor tyrosine kinases but the extracellular cues and upstream intermediates required for activation of the p38 MAPK pathway in myoblast differentiation are not known. Initial observations suggested a correlation between p38 MAPK phosphorylation and cell density, which was also related to N-cadherin levels and Igf2 expression. Subsequent studies using N-cadherin ligand, dominant-negative N-cadherin, constitutively active and dominant-negative forms of RhoA, and MKK6 and p38 constructs, reveal a novel pathway in differentiating myoblasts that links cell-cell adhesion via N-cadherin to Igf2 expression (assessed using northern and promoter-reporter analyses) via RhoA and p38alpha and/or beta but not gamma. We thus define a regulatory mechanism for p38 activation that relates cell-cell-derived adhesion signalling to the synthesis of the major fetal growth factor, IGF-II.

Cadherin adhesion depends on a salt bridge at the N-terminus.

There is now considerable evidence that cell adhesion by cadherins requires a strand exchange process in which the second amino acid at the N-terminus of the cadherin molecule, Trp2, docks into a hydrophobic pocket in the domain fold of the opposing cadherin. Here we show that strand exchange depends on a salt bridge formed between the N-terminal amino group of one cadherin molecule and the acidic side chain of Glu89 of the other. Prevention of this bond in N-cadherin by introducing the mutation Glu89Ala or by extending the N-terminus with additional amino acids strongly inhibited strand exchange. But when the two modifications were present in opposing cadherin molecules respectively, they acted in a complementary manner, lowering activation energy for strand exchange and greatly increasing the strength of the adhesive interaction. N-cadherin that retained an uncleaved prodomain or lacked Trp2 adhered strongly to the Glu89Ala mutant but not to wild-type molecules. Similarly, N-cadherin in which the hydrophobic acceptor pocket was blocked by an isoleucine side chain adhered to a partner that had an extended N-terminus. We explain these results in terms of the free energy changes that accompany strand exchange. Our findings provide new insight into the mechanism of adhesion and demonstrate the feasibility of greatly increasing cadherin affinity.

The mechanism of cell adhesion by classical cadherins: the role of domain 1.

The mechanism by which classical cadherins mediate cell adhesion and, in particular, the roles played by calcium and Trp2, the second amino acid in the N-terminal domain, have long been controversial. We have used antibodies to investigate the respective contributions of Trp2 and calcium to the stability of the N-terminal domain of N-cadherin. Using a peptide antibody to the betaB strand in domain 1, which detects a disordered structure, we show that both Trp2 and calcium play crucial parts in regulating stability of the domain. The epitope for another antibody, mAb GC4, has been mapped to the base of domain 1. Binding of GC4 to this epitope was shown to depend on intramolecular 'docking' of Trp2 into the domain 1 structure. Using this property, we provide evidence that calcium regulates a dynamic equilibrium between docked and undocked Trp2. Finally, a novel technique has been developed to test whether Trp2 cross-intercalation between cadherin molecules from adjacent cells (strand exchange) is central to cadherin-mediated cell adhesion. Guided by crystal structures showing strand exchange, we have introduced single cysteine point mutations into N-cadherin domain 1 in such a way that a disulphide bond will form between opposing N-cadherin molecules during cell adhesion if strand exchange occurs. The bond requires complementary cysteines to be precisely juxtaposed according to the strand exchange model. Our results demonstrate that the disulphide bond forms as predicted. This provides compelling evidence that strand exchange is indeed a primary event in cell adhesion by classical cadherins.

Role of the alphaI domain in ligand binding by integrin alphaEbeta7.

The I domain of integrin alphaE was modeled on the crystal structure of that in CD11b and mutated to produce an open (high affinity) or closed (low affinity) conformation. K562 transfectants expressing mutant alphaE and wild-type beta7 were tested for adhesion to E-cadherin-Fc. Downward displacement of the C terminus of the alphaI domain with a disulfide bridge enhanced adhesion and Mn(2+) dependency. Adhesion greatly exceeded that observed using wild type integrin under similar conditions. The closed integrin gave poor adhesion which was greatly improved by PMA-induced clustering. Blocking beta7 function with a betaI domain-specific antibody inhibited the wild-type but not the locked open integrin. Isolated open alphaI domain expressed on K562 cells showed strong Mn(2+)-dependent adhesion to E-cadherin, whereas the wild-type version was ineffective. alphaEbeta7 was shown to bind to monomeric E-cadherin but to only one component of dimeric E-cadherin. Finally, we report that M290, a function-blocking antibody, bound to a conformation-sensitive epitope near the rim of the alphaI domain MIDAS and recognized wild-type and closed alphaI domain but not the open conformation. The results broadly support the paradigm for affinity regulation by conformational change that has been established for beta2 integrins. Nevertheless, for alphaE, the fully open conformation may represent an extreme situation that does not occur physiologically.

A novel type of deubiquitinating enzyme.

A previous report from this laboratory described two novel proteins that have sequence similarity to A20, a negative regulator of NF-kappaB (Evans, P. C., Taylor, E. R., Coadwell, J., Heyninck, K., Beyaert, R., and Kilshaw, P. J. (2001) Biochem. J. 357, 617-623). One of these molecules, cellular zinc finger anti-NF-kappaB (Cezanne), a 100-kDa cytoplasmic protein, also suppressed NF-kappaB. Here we demonstrate that Cezanne is a novel deubiquitinating enzyme, distinct from the two known families of deubiquitinases, Types I and II. We show that Cezanne contains an N-terminal catalytic domain that belongs to the recently discovered ovarian tumor protein (OTU) superfamily, a group of proteins displaying structural similarity to cysteine proteases but having no previously described function. Recombinant Cezanne cleaved ubiquitin monomers from linear or branched synthetic ubiquitin chains and from ubiquitinated proteins. Mutation of a conserved cysteine residue in the catalytic site of the proteolytic domain caused Cezanne to co-precipitate polyubiquitinated cellular proteins. We also provide evidence for an additional ubiquitin binding site in the C-terminal part of the molecule. Our data provide the first demonstration of functional activity among OTU proteins.