The Helix aspersa (brown garden snail) allergen repertoire.

BACKGROUND: Ingestion of snails can induce strong asthmatic or anaphylactic responses, mainly in house-dust-mite-sensitized patients. The aim of this study was to identify the Helix aspersa (Hel a), Theba pisana (The p) and Otala lactea (Ota l) allergens and the extent of their cross-reactivity with the Dermatophagoides pteronyssinus (Der p) mite. PATIENTS AND METHODS: In 60 atopic patients, skin prick tests (SPT) to snail and D. pteronyssinus, total and specific IgE, specific IgE immunoblots, RAST and immunoblot inhibition assays were performed. RESULTS: Mean total IgE was >1,000 kU/l. Mean specific IgE (class 6 for Der p and class 2 for Hel a) SPT were positive in 44 patients for snail and in 56 for mite. Isoelectric focusing (IEF) and SDS-PAGE followed by immunoblotting of H. aspersa extract enabled the identification of 27 and 20 allergens, respectively. Myosin heavy chains from snails (molecular weight >208 kDa) disclosed two major allergens. Hel a and Der p RAST were strongly inhibited by their homologous extracts, with Hel a RAST being inhibited by the Der p extract to a much greater extent (72.6%) than the inverse (5.6%). A complete inhibition of the immunoblots by their homologous extract was obtained. However, Hel a extract did not inhibit Der p IEF separated recognition. On the other hand, mite extract extensively inhibited snail immunoblots from both IEF and SDS-PAGE separations. Immune detection on chicken, pig, rabbit, cow and horse myosins did not reveal any IgE cross recognition with snail. CONCLUSIONS: In most cases of snail allergy, mite appeared to be the sensitizing agent. Nevertheless, snails may also be able to induce sensitization by themselves. This hypothesis is supported by the finding of specific IgE to Hel a in 2 patients who did not show specific IgE to Der p, and one of them was suffering from asthma after snail ingestion.

Transthyretin, a new cryptic protease.

Transthyretin (TTR) is a plasma homotetrameric protein that acts physiologically as a transporter of thyroxine (T(4)) and retinol, in the latter case through binding to retinol-binding protein (RBP). A fraction of plasma TTR is carried in high density lipoproteins by binding to apolipoprotein AI (apoA-I). We further investigated the nature of the TTR-apoA-I interaction and found that TTR from different sources (recombinant and plasmatic) is able to process proteolytically apoA-I, cleaving its C terminus after Phe-225. TTR-mediated proteolysis was inhibited by serine protease inhibitors (phenylmethanesulfonyl fluoride, Pefabloc, diisopropyl fluorophosphate, chymostatin, and N(alpha)-p-tosyl-l-phenylala-nine-chloromethyl ketone), suggesting a chymotrypsin-like activity. A fluorogenic substrate corresponding to an apoA-I fragment encompassing amino acid residues 223-228 (Abz-ESFKVS-EDDnp) was used to characterize the catalytic activity of TTR, including optimum reaction conditions (37 degrees C and pH 6.8) and catalytic constant (K(m) = 29 microm); when complexed with RBP, TTR activity was lost, whereas when complexed with T(4), only a slight decrease was observed. Cell lines expressing TTR were able to degrade Abz-ESFKVS-EDDnp 2-fold more efficiently than control cells lacking TTR expression; this effect was reversed by the presence of RBP in cell culture media, therefore proving a TTR-specific proteolytic activity. TTR can act as a novel plasma cryptic protease and might have a new, potentially important role under physiological and/or pathological conditions.

Protein kinase C gamma associates directly with the GluR4 alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate receptor subunit. Effect on receptor phosphorylation.

Ionotropic glutamate receptors mediate the majority of excitatory synaptic transmission in the brain and are thought to be involved in learning and memory formation. The activity of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA)-type glutamate receptors can be regulated by direct phosphorylation of their subunits, which affects the electrophysiological properties of the receptor, and the receptor association with numerous proteins that modulate membrane traffic and synaptic targeting of the receptor. In the present study we investigated the association of protein kinase C (PKC) gamma isoform with the GluR4 AMPA receptor subunit. PKC gamma was co-immunoprecipitated with GluR4 AMPA receptor subunit in rat cerebellum and in cultured chick retina cell extracts, and immunocytochemistry experiments showed co-localization of GluR4 and PKC gamma in cultured chick retinal neurons. Pull-down assays showed that native PKC gamma binds the GluR4 C-terminal membrane-proximal region, and recombinant PKC gamma was retained by GST-GluR4 C-terminal fusion protein, suggesting that the kinase binds directly to GluR4. Furthermore, GST-GluR4 C-terminal protein was phosphorylated on GluR4 Ser-482 by bound kinases, retained by the fusion protein, including PKC gamma. The GluR4 C-terminal segment that interacts with PKC gamma, which lacks the PKC phosphorylation sites, inhibited histone H1 phosphorylation by PKC, to the same extent as the PKC pseudosubstrate peptide 19-31, indicating that PKC gamma bound to GluR4 preferentially phosphorylates GluR4 to the detriment of other substrates. Additionally, PKC gamma expression in GluR4 transfected human embryonic kidney 293T cells increased the amount of plasma membrane-associated GluR4. Our results suggest that PKC gamma binds directly to GluR4, thereby modulating the function of GluR4-containing AMPA receptors.