A growth-dependent post-translational modification of annexin VI.

Annexin VI (p68, 67-kDa calelectrin) is a member of a family of Ca2+/phospholipid-binding proteins, that includes p35 (annexin I) and p36 (annexin II), the major cellular substrates for phosphorylation by the epidermal growth factor receptor and pp60v-src tyrosine kinase activities, respectively. We report here that like annexins I and II, annexin VI is phosphorylated in vivo, but that in contrast, annexin VI phosphorylation is associated with cell growth. In both Swiss 3T3 fibroblasts and human T-lymphoblasts the pattern of phosphorylation followed an almost identical profile. In particular, annexin VI was not phosphorylated in quiescent cells, but was phosphorylated on serine and to a lesser extent threonine, several hours following cell stimulation. Furthermore, annexin VI also incorporated phosphate in a growth-dependent manner, in a form other than a phosphoamino-acid. The phosphate was visualised following acid hydrolysis of immunoprecipitated annexin VI, as part of a complex having high mobility on 2-D thin-layer electrophoresis. The identity of this complex is not known. The results suggest that a post-translational modification other than direct protein phosphorylation may influence the activity of annexin VI and provide evidence linking cell growth with regulation of annexin VI function.

Crystallization and preliminary X-ray crystallographic studies of human placental annexin IV.

Human placental annexin IV, a member of the annexin family of calcium and phospholipid-binding proteins, has been crystallized by the vapour diffusion method in the presence of calcium, using polyethylene glycol 8000. The crystals are orthorhombic, space C222(1), cell dimensions a = 105.4 A, b = 115.7 A, c = 80.7 A and diffract to at least 2.5 A resolution on a synchrotron source.

Crystallization of p68 on lipid monolayers and as three-dimensional single crystals.

Two-dimensional crystals of p68, a Ca2+ -binding protein that has homology with members of the lipocortin/calpactin family, were obtained by interaction with a phospholipid monolayer. By measuring surface pressure at constant surface area, p68 was found to interact in a Ca2+ -dependent manner specifically with phosphatidylethanolamine, less so with phosphatidylserine and not at all with phosphatidylcholine. With dimyristoyl-phosphatidylethanolamine, two-dimensional crystalline arrays were formed. Image analysis of electron micrographs of these crystals, which diffracted to about 50 A, revealed p3 symmetry with a unit cell of about 178 A by 178 A; the protein densities showed a two-domain structure giving a cylindrical molecule of about 100 A by 35 A diameter packed as trimers. Three-dimensional microcrystals obtained without lipid or Ca2+ were suitable for electron microscopy and gave a tetragonal unit cell of about 256 A by 68 A. The implications of these observations on the structure and lipid specificity of p68 binding are discussed.

Purification and identification by amino acid sequence analysis of the subunits of the CD3 antigen of human tonsil T-lymphocytes.

The CD3 antigen was purified from human tonsils by immunoaffinity chromatography and preparative SDS-PAGE; the overall yield was 10%. Amino acid sequence analysis of the separated gamma, delta and epsilon polypeptides revealed that the gamma and epsilon polypeptides were blocked at the N-terminus, whereas the partial N-terminal amino acid sequence for the delta chain was identical to that described by Borst et al. [Nature 312, 455-485 (1984)]. The gamma and epsilon chains were cleaved with formic acid and cyanogen bromide respectively in order to obtain amino acid sequence data. The sequences obtained corresponded exactly to the amino acid sequences deduced from the nucleotide sequences of putative gamma and epsilon cDNA clones.

Alternative splicing gives rise to two forms of the p68 Ca2(+)-binding protein.

The p68 Ca2+ and phospholipid binding protein of the lipocortin/calpactin family appears to exist as two forms. These may be resolved into a closely-spaced polypeptide doublet by SDS-PAGE. The cloning and sequencing of p68 revealed an apparent 18 nucleotide alternative splice sequence, which could account for this observation. We show here that an antiserum directed against a synthetic peptide corresponding to the region containing the splice sequence, recognises only the upper band of the p68 doublet by both immunoprecipitation and Western blotting. These results are consistent with alternative splicing being responsible for the generation of the two forms of p68.

2D crystal forms of annexin IV on lipid monolayers.

Two-dimensional crystalline arrays of annexin IV were generated by interaction of the purified protein with a phospholipid monolayer. Image analysis of electron micrographs of the protein crystals, which diffracted to 3.5 nm respectively, revealed p6 and p3 symmetry. Annexin IV gave two crystal forms with unit cells of 18 x 18 nm and 28 x 28 nm. The former unit cell was similar to a previously described form of annexin VI. The implications of these observations are discussed.

A model of the structure of human annexin VI bound to lipid monolayers.

Annexin VI is an eight repeat member of the annexin family of proteins which are both water soluble and bind to negatively charged phospholipids in a calcium-dependent manner. Here we present a model for annexin VI based on fitting the three-dimensional structure of two annexin V molecules (Huber (1990) EMBO J. 9, 3867-3874) to the two-dimensional stain-excluding density of lipid-bound annexin VI (Newman (1989) J. Mol. Biol. 206, 213-219). Both annexin VI lobes could only be fitted with their convex faces closest to the lipid monolayer. This supports the hypothesis that annexin-lipid binding is mediated by the interaction between calcium bound to the loops protruding from the convex protein surface and phospholipid headgroups.

Expression of HLA system antigens on placenta.

The levels of HLA-A and -B antigens expressed by placenta have been assessed relative to parental and other A and B antigen types that were not shared by the foetus. A purified preparation of placenta plasma membrane was used to estimate the antigen activities. The results indicate that maternally and paternally inherited A and B antigen activities and beta2-microglobulin are expressed to similar extents but at much lower levels than in spleen lymphocytes (less than 5%). The possibility that the amounts detected were caused by contamination with blood or maternal tissue was ruled out. The low levels of A and B antigens may account for the lack of a cellular immune response to the other polymorphic cell surface antigens of the trophoblast. No evidence was obtained for the expression of a significant level of Ia antigens.

Antisera against epitopes resistant to denaturation on T3 (CD3) antigen can detect reactive and neoplastic T cells in paraffin embedded tissue biopsy specimens.

Polyclonal rat antisera, raised against affinity purified CD3 antigen, gave strong immunoenzymatic labelling of T cells in routine paraffin embedded sections, with negligible background staining. The specificity of these reactions was confirmed by staining biopsy specimens from 21 previously phenotyped non-Hodgkin's lymphomas (including 14 of T cell origin and six of B cell origin). It is suggested that the ability of the polyclonal anti-CD3 antisera to detect T cells in paraffin sections is due to the presence in these sera of antibodies against fixation resistant epitopes on CD3 antigen, and that immunisation with purified denatured preparations of other white cell associated antigens may broaden the range of antibodies suitable for the phenotypic analysis of leucocytes in routine histological samples.

Activation of protein kinase C modulates the expression of the T3/T cell antigen receptor complex on human T lymphocytes.

Activators of protein kinase C induced a rapid decrease (within 15 min) in the surface expression of the T3 antigen and T-lymphocyte antigen receptor (Ti) on HPB-ALL cells, and a concomitant phosphorylation of the T3 gamma and delta polypeptides; the gamma chain was more extensively phosphorylated than the delta chain. No phosphorylation of the T3 epsilon chain and the Ti alpha and beta polypeptides was detected. Evidence was obtained that the T3 gamma chain is phosphorylated only on serine residues.