Side reactions in the synthesis of phosphotyrosine-containing peptides

Summary A series of phosphopeptides Tyr(PO₃H₂)-Val-Pro-Xxx-Leu (Xxx=Met, Met(O), Nle, Dab or Cys), derived from the native platelet-derived growth factor- receptor (PDGF-) sequence, has been prepared to study their interaction with the src-homology 2 (SH2) domains of the p85 subunit of PI3 kinase. The phosphopeptides were synthesized using Fmoc methodology incorporating N-Boc dibenzyl-protected phosphotyrosine (Boc-Tyr[PO₃(Bzl)₂]) as the N-terminal amino acid, since the benzyl groups can be removed during resin cleavage with TFA. Only peptides containing methionine were found to exist partially as S-benzyl sulfonium salts after TFA cleavage from the resin. The desired peptide could be obtained from the S-benzyl sulfonium salt by hydrogenolysis.     

Streptococcal erythrogenic toxin type C is not a phosphorylated protein. Description of two different purification procedures and investigation of its phosphorylation state

Abstract Erythrogenic toxin type C (ETC) from different streptococcal group A strains was successively purified by absorption on phenylsepharose, acidic dialysis of the eluate at 40% saturated ammonium sulphate solution, CM-Sepharose chromatography, finally by immunoaffinity chromatography on monoclonal antibodies. Second, after growing of bacteria in the presence of [³²P]orthophosphate to phosphorylate ETC, the ETC was purified with phenylsepharose following immunoaffinity chromatography. The occurrence of phosphoamino acids in the purified ETC was investigated by an immunoassay. No phosphoamino acids could be detected in the ETC molecule. Also after radiolabelling with ³²P it was not possible to demonstrate a radioactive signal. The treatment with alkaline phosphatase has no influence on the mitogenicity or position of ETC in isoelectric focusing. The results obtained led to the conclusion that in contrast to the literature, ETC is not a phosphorylated protein.     

Dimerization of beta B2-crystallin: the role of the linker peptide and the N- and C-terminal extensions.

beta B2- and gamma B-crystallins of vertebrate eye lens are 2-domain proteins in which each domain consists of 2 Greek key motifs connected by a linker peptide. Although the folding topologies of beta B2- and gamma B-domains are very similar, gamma B-crystallin is always monomeric, whereas beta B2-crystallin associates to homodimers. It has been suggested that the linker or the protruding N- and C-terminal arms of beta B2-crystallin (not present in gamma B) are a necessary requirement for this association. In order to investigate the role of these segments for dimerization, we constructed two beta B2 mutants. In the first mutant, the linker peptide was replaced with the one from gamma B (beta B2 gamma L). In the second mutant, the N- and C-terminal arms of 15- and 12-residues length were deleted (beta B2 delta NC). The beta B2 gamma L mutant is monomeric, whereas the beta B2 delta NC mutant forms dimers and tetramers that cannot be interconverted without denaturation. The spectral properties of the beta B2 mutants, as well as their stabilities against denaturants, resemble those of wild-type beta B2-crystallin, thus indicating that the overall peptide fold of the subunits is not changed significantly. We conclude that the peptide linker in beta B2-crystallin is necessary for dimerization, whereas the N- and C-terminal arms appear to be involved in preventing the formation of higher homo-oligomers.     

Models of the serine protease domain of the human antithrombotic plasma factor activated protein C and its zymogen.

Three-dimensional structural analysis of physiologically important serine proteases is useful in identifying functional features relevant to the expression of their activities and specificities. The human serine protease anticoagulant protein C is currently the object of many genetic site-directed mutagenesis studies. Analyzing relationships between its structure and function and between naturally occurring mutations and their corresponding clinical phenotypes would be greatly assisted by a 3-dimensional structure of the enzyme. To this end, molecular models of the protease domain of protein C have been produced using computational techniques based on known crystal structures of homologous enzymes and on protein C functional information. The resultant models corresponding to different stages along the processing pathway of protein C were analyzed for structural and electrostatic differences arising during the process of protein C maturation and activation. The most satisfactory models included a calcium ion bound to residues homologous to those that ligate calcium in the trypsin structure. Inspection of the surface features of the models allowed identification of residues putatively involved in specific functional interactions. In particular, analysis of the electrostatic potential surface of the model delineated a positively charged region likely to represent a novel substrate recognition exosite. To assist with future mutational studies, binding of an octapeptide representing a protein C cleavage site of its substrate factor Va to the enzyme's active site region was modeled and analyzed.     

The pharmacophore of the human C5a anaphylatoxin.

We have determined which amino acids contribute to the pharmacophore of human C5a, a potent inflammatory mediator. A systematic mutational analysis of this 74-amino acid protein was performed and the effects on the potency of receptor binding and of C5a-induced intracellular calcium ion mobilization were measured. This analysis included the construction of hybrids between C5a and the homologous but unreactive C3a protein and site-directed mutagenesis. Ten noncontiguous amino acids from the structurally well-defined 4-helix core domain (amino acids 1-63) and the C-terminal arginine-containing tripeptide were found to contribute to the pharmacophore of human C5a. The 10 mostly charged amino acids from the core domain generally made small incremental contributions toward binding affinity, some of which were independent. Substitutions of the C-terminal amino acid Arg 74 produced the largest single effect. We also found the connection between these 2 important regions to be unconstrained.     

Reversible unfolding of fructose 6-phosphate, 2-kinase:fructose 2,6-bisphosphatase.

Reversible unfolding of rat testis fructose 6-phosphate,2-kinase:fructose 2,6-bisphosphatase in guanidine hydrochloride was monitored by following enzyme activities as well as by fluorescence methodologies (intensity, emission maximum, polarization, and quenching), using both intrinsic (tryptophan) and extrinsic (5((2-(iodoacetyl)amino) ethyl)naphthalene-1-sulfonic acid) probes. The unfolding reaction is described minimally as a 4-state transition from folded dimer-->partially unfolded dimer-->monomer-->unfolded monomer. The partially unfolded dimer had a high phosphatase/kinase ratio due to preferential unfolding of the kinase domain. The renaturation reaction proceeded by very rapid conversion (less than 1 s) of unfolded monomer to dimer, devoid of any enzyme activity, followed by slow (over 60 min) formation of the active enzyme. The recovery rates of the kinase and the phosphatase were similar. Thus, the refolding appeared to be a reversal of the unfolding pathway involving different forms of the transient dimeric intermediates. Fluorescence quenching studies using iodide and acrylamide showed that the tryptophans, including Trp-15 in the N-terminal peptide, were only slightly accessible to iodide but were much more accessible to acrylamide. Fructose 6-phosphate, but not ATP or fructose 2,6-bisphosphate, diminished the iodide quenching, but all these ligands inhibited the acrylamide quenching by 25%. These results suggested that the N-terminal peptide (containing a tryptophan) was not exposed on the protein surface and may play an important role in shielding other tryptophans from solvent.     

Self-splicing group I and group II introns encode homologous (putative) DNA endonucleases of a new family.

A new family of protein domains consisting of 50-80 amino acid residues is described. It is composed of nearly 40 members, including domains encoded by plastid and phage group I introns; mitochondrial, plastid, and bacterial group II introns; eubacterial genomes and plasmids; and phages. The name "EX1HH-HX3H" was coined for both domain and family. It is based on 2 most prominent amino acid sequence motifs, each encompassing a pair of highly conserved histidine residues in a specific arrangement: EX1HH and HX3H. The "His" motifs often alternate with amino- and carboxy-terminal motifs of a new type of Zn-finger-like structure CX2,4CX29-54[CH]X2,3[CH]. The EX1HH-HX3H domain in eubacterial E2-type bacteriocins and in phage RB3 (wild variant of phage T4) product of the nrdB group I intron was reported to be essential for DNA endonuclease activity of these proteins. In other proteins, the EX1HH-HX3H domain is hypothesized to possess DNase activity as well. Presumably, this activity promotes movement (rearrangement) of group I and group II introns encoding the EX1HH-HX3H domain and other gene targets. In the case of Escherichia coli restrictase McrA and possibly several related proteins, it appears to mediate the restriction of alien DNA molecules.     

Monte Carlo study of the effect of β2-microglobulin on the binding cleft of the HLA-A2 complex

Peptide recognition by class I products of the major histocompatibility complex requires association of the class I heavy chain with β₂-microglobulin. We present results of Monte Carlo simulations of the β-pleated sheet floor of the human class I MHC molecule, HLA-A2, with and without β₂-microglobulin. We find a significant effect of β₂-microglobulin on the side chains of residues near a region that would accommodate the C-terminus of a bound peptide. By modeling simultaneously each loop and its neighboring strand at either end of the class I cleft, we find that β₂-microglobulin restricts the conformational space of residues that are central to binding peptides. The effect is most pronounced for R97 and H114 and somewhat less important for Y99 and Y116, the latter forming strong hydrogen bonds with neighboring residues in the heavy chain itself.     

Ectopic expression of β-lactoglobulin/human serum albumin fusion genes in transgenic mice: hormonal regulation andin situ localization

We produced transgenic mice carrying the native sheep -lactoglobulin (BLG) or fusion genes composed of the BLG promoter and human serum albumin (HSA) minigenes. BLG was expressed exclusively in the mammary glands of the virgin and lactating transgenic mice evaluated. In contrast, transgenic females carrying the BLG/HSA fusion constructs also expressed the HSA RNA ectopically in skeletal muscle, kidney, brain, spleen, salivary gland and skin. Ectopic expression of HSA RNA was detected only in strains that express the transgene in the mammary gland. There was no obvious correlation between the level of the HSA RNA expressed in the mammary gland and that found ectopically. In three transgenic strains analysed, the expression of HSA RNA in kidney and skeletal muscle increased during pregnancy and lactation, whereas in the brain HSA expression decreased during lactation in one of the strains. HSA protein was synthesized in skeletal muscle and skin of strain #23 and its level was higher in lactating mice compared with virgin mice. Expression of HSA was also analysed in males and was found to be more stringently controlled than in females of the same strains.In situ hybridization analyses localized the expressed transgene in the skin, kidney, brain and salivary glands of various transgenic strains. Distinct strain-specific and cell-type specific HSA expression patterns were observed in the skin. This is in contrast to the exclusive expression of the HSA transgene in epithelial cells surrounding the alveoli of the mammary gland. Taken together, these results suggest that the absence of sufficient mammary-specific regulatory elements in the BLG promoter sequences and/or the juxtaposition of the BLG promoter with the HSA coding sequences leads to novel tissue- and cell-specific expression in ectopic tissues of transgenic mice.     

Relationship between soluble tumor necrosis factor (TNF) receptors and TNFα during immunotherapy with interleukin-2 and/or interferon α

Eleven metastatic cancer patients were studied during three different regimens of immunotherapy with interleukin-2 (IL-2) and/or interferon (IFN): group A received 4 days of IL-2 i.a. infusion (n=3), group B IFN s.c. during 5 days (n=4), followed on day 3 by 5 days of a continuous IL-2 i.v. infusion, and group C had 4 days of IL-2 i.v. infusion together with s.c. IFN on days 1 and 4 (n=4). Soluble tumor necrosis factor receptors (sTNFR) p55 and p75 and TNF concentrations in serum were analyzed before therapy and daily during 8 days of the first therapy cycle. sTNFR was measured by radioimmunoassay. sTNFR p55 increased in all patient groups from a baseline value of 5.2±0.9 ng/ml to a maximum of 13.6±1.2 ng/ml by days 3–4 (P=0.003). sTNFR p75 increased from 7.6±1.1 ng/ml to peak values of 30.1±2.6 ng/ml in groups A and B (P=0.02). In group C the sTNFR p75 response was weak (NS). In group B, the increase of both p55 and p75 occurred only after addition of IL-2 to IFN. TNF increased weakly during treatment with IFN alone (group B); it rose strongly during IL-2 and the combined treatment (groups A-C) from 8±2 pg/ml to 115±13 pg/ml (P=0.003). In group B, it reached the maximum 24 h after addition of IL-2 to IFN and decreased thereafter. there was a significant relationship between TNF and sTNFR p55 or sTNFR p75 in groups A and C, (P=0.001), but not in group B. Group C was also investigated during the third therapy cycle. The increase of sTNFR p75 was stronger (P=0.01) and that of TNF weaker than in the first cycle; the sTNFR p55 response was similar in both cycles. In conclusion sTNFR p55 and p75 are rapidly induced during IL-2 and IL-2+IFN treatment, the increase of sTNF receptors parallels or exceeds that of TNF and may influence the immunomodulatory effects of TNF during cytokine therapy.