Thomas Vorherr

H+-induced membrane insertion of influenza virus hemagglutinin involves the HA2 amino-terminal fusion peptide but not the coiled coil region.

Fusion of influenza virus with target membranes is induced by acid and involves complex changes in the viral envelope protein hemagglutinin (HA). In a first, kinetically distinct step, the HA polypeptide chain 2 (HA2) is inserted into the target membrane bilayer. Using hydrophobic photolabeling with the phospholipid analogue 1-O-hexadecanoyl-2-O-[9-[[[2-[125I]iodo-4(trifluoromethyl-3H-diazirin-3-yl)benzyl]oxy]carbonyl]nonanoyl]-sn-glycero-3-phosphocholine, we identified the segment within HA2 that interacts with the membrane. The sole part of the HA2 ectodomain that was labeled with the membrane-restricted reagent is the NH2-terminal fusion peptide (residues 1-22). No labeling occurred within the long coiled coil region generated during the acid-induced conformational transition (Bullough, P. A., Hughson, F. M., Skehel, J. J., and Wiley, D. C. (1994) Nature 371, 37-43). These data strongly suggest that the coiled coil region of HA2 does not insert into the lipid bilayer. This conclusion is at variance with the recent suggestion (Yu, Y. G., King, D. S., and Shin, Y.-K. (1994) Science 266, 274-276) that the coiled coil of HA may splay apart and insert into the target membrane, providing a mechanism by which the viral and the target membrane may come in close apposition.

Differential and multiple binding of signal transducing molecules to the ITAMs of the TCR‐ζ chain

A biotin‐streptavidin‐based technique was developed for high affinity, unidirectional, and specific immobilization of synthetic peptides to a solid phase. Biotinylated 23‐mer carboxamide peptides corresponding to the three immunoreceptor tyrosine‐based activation motifs (ITAMs) of the T cell antigen receptor associated ζ‐chain (TCR‐ζ) in their bis‐, mono‐, or unphosphorylated forms were used to study the binding of cellular proteins from human Jurkat T cells to these signal transduction motifs. The protein tyrosine kinase ZAP‐70 bound specifically to all bisphosphorylated peptides but not to the mono‐ or unphosphorylated peptides. In contrast, Shc, phosphatidylinositol 3‐kinase (P13K), Grb2, and Ras‐GTPase activating protein (GAP) bound with different affinities to the bis‐ or monophosphorylated peptides, while the Src family protein tyrosine kinase (PTK) Fyn did not bind specifically to any of the tested peptides. The different preferences of the studied signaling molecules for distinct ITAMs, and in particular the binding of some of them preferentially to monophosphorylated peptides, suggests that the TCR‐ζ may bind multiple signaling molecules with each ITAM binding a unique set of such molecules. In addition, partial phosphorylation of the ITAMs may result in recruitment of different proteins compared to double phosphorylation. This may be crucial for coupling of the TCR to various effector functions under different conditions of receptor triggering.

Synthesis and application of an auxiliary group for chemical ligation at the X-gly site

An efficient synthesis of an auxiliary group, the 2-mercapto-4,5-dimethoxybenzyl (Dmmb) moiety, to form a Gly-building block is presented. The building block was incorporated into peptides to study the reaction with thiobenzyl-activated derivatives. The target peptides have been obtained by standard chemical ligation reaction, followed by TMSBr-assisted cleavage to remove the auxiliary group. Prior to Dmmb removal, under acidic conditions an unexpected rearrangement was observed and evidence for a mechanism is provided.

Phospho-serine and phospho-threonine building blocks for the synthesis of phosphorylated peptides by the Fmoc solid phase strategy

Abstract The synthesis of phospho-Ser and phospho-Thr-containing peptides based on Nα-Fmoc-O-(monobenzylphosphono)-Ser and -Thr building blocks is described. The building blocks were obtained by phosphorylation of the Fmoc-protected allyl ester derivatives following oxidation, removal of the allyl-group and selective cleavage of one phospho benzyl ester function with NaI.

GLOBAL PHOSPHORYLATION OF PEPTIDES CONTAINING OXIDATION-SENSITIVE AMINO ACIDS

Abstract We report on an extension of the global phosphorylation procedure for the preparation of phosphopeptides containing oxidation-sensitive amino acids such as tryptophan, methionine or cysteine.

Bachem, a Swiss speciality cGMP-manufacturer: History and future prospects

This article summarizes the development of the Bachem Group and the ongoing commitment regarding its production facilities in Switzerland. Bachems core business, the manufacture of peptides from lab scale to market quantities, is explained using showcases to detail the high level of know-how needed to successfully prepare very large biomolecules. Based on these examples, the complexity regarding production processes and allocation of manufacturing sites are elucidated.

S8.5 The fourth immunoglobulin-like domain of NCAM contains a carbohydrate recognition domain for oligomannosidic glycans implicated in association with LI and neurite outgrowth

In the 3-D structure of Erythrina corallodendron lectin (ECorL) in complex with a ligand (lactose), the first of its kind for a Gal/GalNAc-specific lectin (Shaanan et al., 1991, Science, 254, 862), a hydrophobic cavity is present surrounded by Tyr I°s, Pro TM and Trp 13~ not in contact with Gal of the lactose in the combining site. The cavity can accommodate bulky substituents such as acetamido or dansylamido (NDns) at C-2 of the lectin-bound Gal. Molecular modelling suggested that the Dns of MeflGalNDns, which binds the lectin ~300 times stronger than Gal, fits snugly into the cavity. Comparison of the primary sequence of ECorL with that of the homologous peanut agglutinin (PNA), which is Gal specific but does not bind GalNAc or GalNDns, showed that in PNA such a cavity is absent, being filled by two extra amino acids. We therefore constructed a mutant L2 in which Pro134-Trp 135 in ECorL was replaced by Ser-Glu-Tyr-Asn (as in PNA), a single point mutant Y108T (also as in PNA), a double mutant L2;Y108T, and another single point mutant W135A. They were expressed in Escherichia coli as done for rECorL (Arango et al., 1992, Eur. J. Biochem., 205, 575) and examined for their specificity by inhibition of hemagglutination and of binding of the tectin to asialofetuin. In the latter assay, Y108T had a specificity similar to rECorL; L2 had the same affinity for Gal as rECorL, and 8 and 15 times lower affinity for GalNAc and MeflGalNDns, respectively. L2;Y108T had a similar affinity for Gal and GalNDns as L2, but twice the affinity for GalNAc. W135A, on the other hand, bound Gal 5 times stronger, and GalNAc and GalNDns 3 and 6 times weaker, respectively, than rECorL. Our results demonstrate that (a) Trp m contributes to the strong binding of MeflGalNDns to ECorL, whereas Tyr l°s does not; (b) the region close to Trp ~35 must be free in order to allow the lectin to bind Gal derivatives with bulky substituents at C-2; (c) changing a residue such as Trp ~35, which is not in direct contact with the Gal ligand, may change the conformation of the combining site of the lectin.