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.     

Mass spectral characterization of a protein-nucleic acid photocrosslink

A photocrosslink between basic fibroblast growth factor (bFGF155) and a high affinity ssDNA oligonucleotide was characterized by positive ion electrospray ionization mass spectrometry (ESIMS). The DNA was a 61-mer oligonucleotide photoaptamer bearing seven bromodeoxyuridines, identified by in vitro selection. Specific photocrosslinking of the protein to the oligonucleotide was achieved by 308 nm XeCl excimer laser excitation. The cross-linked protein nucleic acid complex was proteolyzed with trypsin. The resulting peptide crosslink was purified by PAGE, eluted, and digested by snake venom phosphodiesterase/alkaline phosphatase. Comparison of the oligonucleotide vs. the degraded peptide crosslink by high performance liquid chromatography coupled to an electrospray ionization triple quadrupole mass spectrometer showed a single ion unique to the crosslinked material. Sequencing by collision induced dissociation (MS/MS) on a triple quadrupole mass spectrometer revealed that this ion was the nonapeptide TGQYKLGSK (residues 130-138) crosslinked to a dinucleotide at Tyr133. The MS/MS spectrum indicated sequential fragmentation of the oligonucleotide to uracil covalently attached to the nonapeptide followed by fragmentation of the peptide bonds. Tyr133 is located within the heparin binding pocket, suggesting that the in vitro selection targeted this negative ion binding region of bFGF155.     

A robust and cost-effective method for the production of Val, Leu, Ile (δ1) methyl-protonated 15N-, 13C-, 2H-labeled proteins

A selective protonation strategy is described that uses [3-2H] 13C -ketoisovalerate to introduce (1H- methyl)-leucine and (1H- methyl)-valine into 15N-, 13C-, 2H-labeled proteins. A minimum level of 90% incorporation of label into both leucine and valine methyl groups is obtained by inclusion of 100 mg/L -ketoisovalerate in the bacterial growth medium. Addition of [3,3-2H2] -ketobutyrate to the expression media (D2O solvent) results in the production of proteins with (1H-1 methyl)-isoleucine (>90% incorporation). 1H-13C HSQC correlation spectroscopy establishes that CH2D and CHD2 isotopomers are not produced with this method. This approach offers enhanced labeling of Leu methyl groups over previous methods that utilize Val as the labeling agent and is more cost effective.     

Ontogeny of the opioidergic regulation of LH and prolactin secretion in lactating sows. II: Interaction between suckling and morphine administration

Administration of morphine to ten suckled and nine zero-weaned (piglets removed immediately after farrowing) sows was used to investigate the apparent absence of opioid regulation of LH and prolactin secretion in early lactation. Blood samples were collected at 10 min intervals at 24-30, 48-54, 72-78 h post partum, and for a 12 h period from 08:00 to 20:00 on day 10 after farrowing. Morphine (0.1 mg kg-1) was administered as three i.v. bolus injections at intervals of 1 h during the last 3 h of each of the 6 h sampling periods, and at 6, 7 and 8 h after the beginning of sampling on day 10. There were significant (P < 0.001) group (zero-weaned versus suckled), time and morphine effects on LH secretion. Plasma LH concentrations increased (P < 0.001) within 48 h of farrowing in zero-weaned sows. Long-term trends of an increase in mean plasma LH in the sampling periods before treatment were attenuated in both groups by morphine treatment. Morphine also significantly inhibited (P < 0.05) prolactin secretion in suckled sows. In zero-weaned sows, plasma prolactin was already low at the start of sampling and did not change with time or in response to morphine treatment. Therefore, the inability to demonstrate an opioidergic involvement in the suckling-induced inhibition of LH secretion during the early post-partum period in sows is not due to a lack of opioid receptors. Furthermore, in suckled sows, morphine is stimulatory to systems that have an inhibitory effect on prolactin secretion.     

Function of herpes simplex virus type 1 gD mutants with different receptor-binding affinities in virus entry and fusion

We have studied the receptor-specific function of four linker-insertion mutants of herpes simplex virus type 1 glycoprotein D (gD) representing each of the functional regions of gD. We used biosensor analysis to measure binding of the gD mutants to the receptors HVEM (HveA) and nectin-1 (HveC). One of the mutants, gD(inverted Delta 34t), failed to bind HVEMt but showed essentially wild-type (WT) affinity for nectin-1t. The receptor-binding kinetics and affinities of the other three gD mutants varied over a 1,000-fold range, but each mutant had the same affinity for both receptors. All of the mutants were functionally impaired in virus entry and cell fusion, and the levels of activity were strikingly similar in these two assays. gD(inverted Delta 34)-containing virus was defective on HVEM-expressing cells but did enter nectin-1-expressing cells to about 60% of WT levels. This showed that the defect of this form of gD on HVEM-expressing cells was primarily one of binding and that this was separable from its later function in virus entry. gD(inverted Delta 243t) showed WT binding affinity for both receptors, but virus containing this form of gD had a markedly reduced rate of entry, suggesting that gD(inverted Delta 243) is impaired in a postbinding step in the entry process. There was no correlation between gD mutant activity in fusion or virus entry and receptor-binding affinity. We conclude that gD functions in virus entry and cell fusion regardless of its receptor-binding kinetics and that as long as binding to a functional receptor occurs, entry will progress.     

"Soft"-cuticle protein secondary structure as revealed by FT-Raman, ATR FT-IR and CD spectroscopy.

The nature of the interaction of insect cuticular proteins and chitin is unknown even though about half of the cuticular proteins sequenced thus far share a consensus region that has been predicted to be the site of chitin binding. We previously predicted the preponderance of a beta-pleated sheet in the consensus region and proposed its responsibility for the formation of helicoidal cuticle (Iconomidou et al., Insect Biochem. Mol. Biol. 29 (1999) 285). In this study, we examined experimentally the secondary structure of intact and guanidine hydrochloride extracted cuticle and the cuticular protein extract. The studied cuticle came from the larval dorsal abdomen of the lepidopteran Hyalophora cecropia, a classical example of "soft" cuticle. Analysis with FT-Raman, ATR FT-IR and CD spectroscopy indicates that antiparallel beta-pleated sheet is the predominant molecular conformation of "soft-cuticle" proteins both in situ in the cuticle and following extraction. It seems that this conformation dictates the modes of chitin-protein interaction in cuticle, in agreement with earlier proposals (Atkins, J. Biosci. 8 (1985) 375).     

Minor quantitative trait loci underlie floral traits associated with mating system divergence in Mimulus

The genetic basis of species differences provides insight into the mode and tempo of phenotypic divergence. We investigate the genetic basis of floral differences between two closely related plant taxa with highly divergent mating systems, Mimulus guttatus (large-flowered outcrosser) and M. nasutus (small-flowered selfer). We had previously constructed a framework genetic linkage map of the hybrid genome containing 174 markers spanning approximately 1800 cM on 14 linkage groups. In this study, we analyze the genetics of 16 floral, reproductive, and vegetative characters measured in a large segregating M. nasutus x M. guttatus F2 population (N = 526) and in replicates of the parental lines and F1 hybrids. Phenotypic analyses reveal strong genetic correlations among floral traits and epistatic breakdown of male and female fertility traits in the F2 hybrids. We use multitrait composite interval mapping to jointly locate and characterize quantitative trait loci (QTLs) underlying interspecific differences in seven floral traits. We identified 24 floral QTLs, most of which affected multiple traits. The large number of QTLs affecting each trait (mean = 13, range = 11-15) indicates a strikingly polygenic basis for floral divergence in this system. In general, QTL effects are small relative to both interspecific differences and environmental variation within genotypes, ruling out QTLs of major effect as contributors to floral divergence between M. guttatus and M. nasutus. QTLs show no pattern of directional dominance. Floral characters associated with pollinator attraction (corolla width) and self-pollen deposition (stigma-anther distance) share several pleiotropic or linked QTLs, but unshared QTLs may have allowed selfing to evolve independently from flower size. We discuss the polygenic nature of divergence between M. nasutus and M. guttatus in light of theoretical work on the evolution of selfing, genetics of adaptation, and maintenance of variation within populations.