Anthony C. Willis

Rapid histone H3 phosphorylation in response to growth factors, phorbol esters, okadaic acid, and protein synthesis inhibitors

When quiescent cells are stimulated with growth factors, phorbol esters, okadaic acid, or protein synthesis inhibitors, the early-response genes, which include c-fos and c-jun, are rapidly induced. The earliest growth factor- and phorbol ester-stimulated nuclear signaling events concomitant with proto-oncogene induction are the rapid phosphorylation of two chromatin-associated proteins, pp33 and pp15. We show here that the tumor promoter okadaic acid, which inhibits protein phosphatases 1 and 2A, and the protein synthesis inhibitors anisomycin and cycloheximide also stimulate pp33 and pp15 phosphorylation. Using transcriptional inhibitors, we show that this response is not a consequence of early gene induction. By peptide mapping and microsequencing, chromatin-associated pp15 is identified as histone H3. Upon stimulation, histone H3 is rapidly phosphorylated on serine residues within its highly charged, basic N-terminal domain. Thus, these diverse agents elicit a common early nuclear signal modulating nucleosomal structure or function, potentially contributing to conformational regulation of proto-oncogene induction.

MASP-3 and Its Association with Distinct Complexes of the Mannan-Binding Lectin Complement Activation Pathway

The mannan-binding lectin (MBL) pathway of complement activation is part of the innate immune defense. The binding of MBL to microbial carbohydrates activates the MBL-associated serine proteases (MASPs), which recruit the complement factors, C4 and C2, to generate the C3 convertase or directly activate C3. We present a phylogenetically highly conserved member of the MBL complex, MASP-3, which is generated through alternative splicing of the MASP-1/3 gene. The designation of MASP-3 as a protease is based on homology to known MASPs. Different MBL oligomers were found to have distinct MASP composition and biological activities. MASP-1, MAp19, and direct C3-cleaving activity are associated with smaller oligomers whereas MASP-3 is found together with MASP-2 on larger oligomers. MASP-3 downregulate the C4 and C2 cleaving activity of MASP-2.

Differentiation inhibiting activity is produced in matrix-associated and diffusible forms that are generated by alternate promoter usage

The differentiation of embryonic stem (ES) cells is controlled by the regulatory factor differentiation inhibiting activity/leukemia inhibitory factor (DIA/LIF). Examination of feeder cell-mediated suppression of ES cell differentiation revealed that DIA/LIF is produced both as a diffusible protein and in an immobilized form associated with the extracellular matrix. This alternative localization arises from the expression of alternate transcripts that diverge throughout exon 1. The effect of alternate first exon usage is to change the amino terminus of the primary translation product and to direct incorporation of mature, biologically active DIA/LIF into the extracellular matrix. The production of a potent regulatory factor in both diffusible and immobilized forms may be an important element of developmental control mechanisms.

Sialoadhesin, a macrophage sialic acid binding receptor for haemopoietic cells with 17 immunoglobulin-like domains

Sialoadhesin is a macrophage‐restricted adhesion molecule of 185 kDa that mediates sialic acid‐dependent binding to cells. It is expressed strongly by macrophages in lymphoid and haemopoietic tissues where it is likely to mediate cell‐cell interactions. Here we report the molecular cloning of murine sialoadhesin and show that it is a new member of the immunoglobulin (Ig) superfamily with 17 Ig‐like domains. COS cells transfected with a cDNA encoding full‐length sialoadhesin bound mouse bone marrow cells in a sialic acid‐dependent manner. Alternatively spliced cDNAs, predicting soluble forms of sialoadhesin containing the first three or 16 Ig‐like domains of sialoadhesin, were expressed in COS cells and the respective proteins purified. When immobilized on plastic, the 16‐domain form bound cells in a sialic acid‐dependent manner, suggesting that sialoadhesin can function in both secreted and membrane‐bound forms. The most similar proteins in the database were CD22, myelin‐associated glycoprotein, Schwann cell myelin protein and CD33. Like sialoadhesin, CD22 mediates sialic acid‐dependent cell adhesion. The sequence similarity of sialoadhesin to CD22 and related members of the Ig superfamily indicates the existence of a novel family of sialic acid binding proteins involved in cell‐cell interactions.

Complement inhibitor of C5 activation from the soft tick Ornithodoros moubata

Blood-feeding ticks must control C activation or be damaged by the host inflammatory response. We report the characterization and expression of a novel, relatively small, broad-acting C inhibitory protein (termed OmCI) from the soft tick Ornithodoros moubata. The native 17-kDa nonglycosylated protein inhibits both human and guinea pig classical and alternative C activation pathways. The IC50 values for each pathway were 12 and 27 nM, respectively, in hemolytic assays using human serum diluted 40-fold. The cDNA encodes a protein of 168 aa, including an 18-aa secretion signal sequence that is absent in the mature form. The inhibitor has 46% amino acid identity with moubatin, a platelet aggregation inhibitor also from O. moubata that is an outlying member of the lipocalin family. Native OmCI had no inhibitory effect on the addition of C8 and C9 to preformed C5b-C7 and C5b-C8 to form the membrane attack complex and no effect on the rate of C3a production by the C3 convertase enzymes C4bC2a, C3(H2O)Bb, or C3bBb. Both recombinant and native OmCI abolish production of C5a by human classical (C4bC3bC2a) and alternative (C3bC3bBb) C5 convertases. Addition of excess C5 but not C3 competes away the inhibitory activity of OmCI, indicating that OmCI targets C5 itself rather than inhibiting the C5 convertase C4bC3bC2a itself. Direct binding of OmCI to C5 was demonstrated by Western blotting and gel filtration chromatography using 125I-labeled proteins. OmCI is the first lipocalin family member shown to inhibit C and also the first natural inhibitor that specifically targets the C5 activation step.

Lymphocyte specific heterogeneity in the rat leucocyte common antigen (T200) is due to differences in polypeptide sequences near the NH2-terminus.

The leucocyte‐common antigen (L‐CA, T200 or CD45) consists of a family of heavily glycosylated glycoproteins of apparent Mr 180,000‐240,000 which are restricted to lymphoid and myeloid cells. Forms of L‐CA which differ in their apparent Mr, antigenicity and glycosylation are expressed on different lymphocyte types. One specific antigenic determinant called MRC OX‐22 is of particular interest because it distinguishes two sets of T helper cells that have different functions. From the sequence of different L‐CA cDNA clones we now conclude that there is sequence heterogeneity such that at least four forms of L‐CA exist with sequences in the range 1118‐1250 amino acids. All the sequence variation occurs at a point starting 6 residues from the NH2‐terminus and the last 1112 residues of all forms are identical. Two of the variants can be directly related to the antigenic variation because they include sequence that was determined for a peptide that carries the MRC OX‐22 determinant. Analysis of glycopeptides from thymocyte L‐CA identified only one non‐glycosylated position out of 14 possible N‐glycosylation sites and established that all O‐glycosylation was within the first 32 amino acids. The extra protein sequence in the longer forms was also suggestive of extensive O‐glycosylation.

Naturally Processed HLA Class II Peptides Reveal Highly Conserved Immunogenic Flanking Region Sequence Preferences That Reflect Antigen Processing Rather Than Peptide-MHC Interactions

MHC class II heterodimers bind peptides 12–20 aa in length. The peptide flanking residues (PFRs) of these ligands extend from a central binding core consisting of nine amino acids. Increasing evidence suggests that the PFRs can alter the immunogenicity of T cell epitopes. We have previously noted that eluted peptide pool sequence data derived from an MHC class II Ag reflect patterns of enrichment not only in the core binding region but also in the PFRs. We sought to distinguish whether these enrichments reflect cellular processes or direct MHC-peptide interactions. Using the multiple sclerosis-associated allele HLA-DR2, pool sequence data from naturally processed ligands were compared with the patterns of enrichment obtained by binding semicombinatorial peptide libraries to empty HLA-DR2 molecules. Naturally processed ligands revealed patterns of enrichment reflecting both the binding motif of HLA-DR2 (position (P)1, aliphatic; P4, bulky hydrophobic; and P6, polar) as well as the nonbound flanking regions, including acidic residues at the N terminus and basic residues at the C terminus. These PFR enrichments were independent of MHC-peptide interactions. Further studies revealed similar patterns in nine other HLA alleles, with the C-terminal basic residues being as highly conserved as the previously described N-terminal prolines of MHC class II ligands. There is evidence that addition of C-terminal basic PFRs to known peptide epitopes is able to enhance both processing as well as T cell activation. Recognition of these allele-transcending patterns in the PFRs may prove useful in epitope identification and vaccine design.

Activity, disulphate mapping and structural modelling of the fifth domain of human β2-glycoprotein I

Complexes formed by the interaction of negatively charged phospholipids and β2‐glycoprotein I(β2‐I) are the target of autoantibodies in systemic lupus erythematosus. The highly positively charged fifth (C‐terminal) domain of human β2‐I was produced as a fusion protein in an Escherichia coli expression system and was shown to bind to the negatively charged phospholipid, cardiolipin, almost as well as the intact protein. In an attempt to define the 3D structure of this domain, the disulphate linkage pattern was determined and shown to be Cys 1–4, Cys 2–5 and Cys 3–6 in contradiction to an earlier report. In the light of this information, the sequence of the fifth domain of β2I(β2‐I‐5) is readily aligned with that of the 16th repeat of factor H, of which the 3D structure is known, and a model of β2I‐5 has been built by homology. On the basis of the model we suggest residues that might be the target of profitable site‐directed mutagenesis in structure—function studies.

Purification and Characterization of Two Mannan-Binding Lectins from Mouse Serum

Mannan-binding lectin (MBL) is a serum protein that activates the complement system after binding to glycoconjugates found on the surface of microorganisms. By molecular cloning two forms of MBL have been identified in the mouse (mMBL-A and mMBL-C), but only mMBL-A has been purified and characterized at the protein level. MBL-C has been termed the liver form of MBL. The present report describes the purification and characterization of mMBL-A and mMBL-C from serum. The two forms of mMBL could be separated both by ion-exchange and carbohydrate-affinity chromatography. The initial identification by immunochemical technique was confirmed by N-terminal amino-acid sequencing. Both proteins give bands corresponding to polypeptide chains of 28 kDa on SDS-PAGE in the reduced state, but mMBL-A migrated more rapidly than mMBL-C in acid/urea-PAGE, in accordance with the calculated pIs. Both forms mediated activation of complement component C4 in mannan-coated microtiter wells. MBL-A showed a higher affinity for d-glucose and α-methyl-d-glucose then did MBL-C. Serum concentrations of mMBL-A in laboratory strains and wild mice were found to vary from 5 to 80 μg/ml, with wild mice tending to show higher levels than laboratory strains.

The MRC OX-45 antigen of rat leukocytes and endothelium is in a subset of the immunoglobulin superfamily with CD2, LFA-3 and carcinoembryonic antigens.

The MRC OX‐45 cell surface antigen is a glycoprotein of 45,000 apparent mol. wt of rat leukocytes and endothelium. Antibodies against the antigen inhibit T lymphocyte responses by stimulation of suppression by accessory cells. We now report the immunochemical characterization of this antigen and its cDNA sequence. The predicted protein sequence contains 240 amino acids including a leader sequence of 22 residues and a carboxy‐terminal sequence of 23 residues that is replaced in the processed molecule by a glycosyl‐phosphatidylinositol anchor attached at serine 195. Two Ig‐related domains are predicted to account for all of the processed sequence and the circular dichroism spectrum shows pure beta‐structure. The amino‐terminal domain is V‐like, but without a disulphide bond, while the second domain is C‐like (C2‐SET) with two disulphide bonds. The sequence matches particularly well with the extracellular parts of LFA‐3 and CD2 antigens and the first two domains of carcinoembryonic antigen and non‐specific, cross‐reacting antigen.