A solid-phase assay for the complete subsite mapping doftheactive site ofeldop eaes has been developed.A lbrary of resin-bound ptase ubstrates was sytsed both on kieselguhr-supported polyamide resin and on a polyethylene glycol-poly-(NN-dlnethylacryhmlde) copolymer type of resin that alows proteas to diffuse into the interior and perform their catalytic activity. An lic acid and 3-it rosine were used a an efclet donor-acceptor p for the resonance energy transfer. The synthesis was performed in a manual library generator that allows simple wet ming of the bead and ll washing p eus. After treatment wit subtiAsin Carlsberg, beads were c and subjected to peptide seq , affording the preferred s quences, their cleavage bond, and a esiqstimation of the turnover. A satitcal dbution of prefed amino acids was obtain for each subsite. The result was compaed with data from kinetic studies In solution.In early studies of the activity of isolated proteolytic enzymes, polypeptides were subjected to digestion with the enzyme (1) and the preferred cleavage site, if any, was determined by isolation of the fragments and subsequent Edman degradation. This often tedious work provided lead amino acid sequences that could be used for determination of the optimal substrates. These were prepared by chemical synthesis and the kinetic constants were measured by determination ofthe rate ofproduct formation by HPLC (2), NMR spectroscopy (3), or spectrophotometric monitoring when chromogenic or fluorogenic substrates could be used (4, 5). More recently the development of internally quenched fluorogenic substrates of the resonance energy transfer type (6, 7) has facilitated the complete subsite mapping (5,(8)(9)(10)(11)(12) for endoproteases. In these substrates, it is important that efficient long-range energy transfer is observed between the donor and the acceptor to span the entire active site of the endopeptidase, thus minimizing the interaction between the enzyme and the chromophoric probes. The donor-acceptor amino acid pair o-aminobenzamide (ABz)-3-nitrotyrosine [Tyr(NO2)] (13) for which excellent quenching of fluorescence is observed has recently been described (5). This donor-acceptor pair is conveniently introduced in parallel multiple-column peptide synthesis (MCPS) (14) of numerous substrates and has been used for subsite mapping of a variety of proteases (6,(15)(16)(17)(18)(19)(20)(21). However, substantial effort is still required to identify the optimal substrates.The use ofcombinatorial peptide libraries (22) and portionmixing libraries (23, 24) is widely accepted as the method of choice for defining binding motifs and unknown biological activities. The portion-mixing library is particularly convenient for the presentation of millions of substrates to a protease with unknown specificity. The problem is, however, to detect, isolate, and characterize the active substances. Due to the high quantum yield of the ABz group, the fluorescence can easily be observed visually in the absence of Tyr(N02), whereas peptides containi...

Three types of beaded polyethylene glycol polyacrylamide copolymers (PEGA) with a high content of polyethylene glycol (PEG) were synthesized by inverse suspension polymerization and characterized for peptide synthesis and with respect to their physical properties. Several peptides of high purity have been synthesized on the resin. The properties which were determined were loading of amino groups, swelling, bead size distribution, porosity, flexibility and compatibility with active biomolecules. A loading of 0.35 mmol/g has been obtained and the swelling was excellent in solvents of various polarities ranging from water to dichloromethane. The 13C-NMR T1-relaxation times of a resin containing a peptide were determined in DMSO-d6 and the resin was found to exhibit a behavior similar to the components in free solution.

No abstract

To investigate the molecular basis of antigenic mimicry by peptides, we studied a panel of closely related mAbs directed against the cell-wall polysaccharide of group A Streptococcus. These antibodies have restricted V-gene usage, indicating a shared mechanism of binding to a single epitope. Epitope mapping studies using synthetic fragments of the cell-wall polysaccharide supported this conclusion. All of the mAbs isolated crossreactive peptides from a panel of phagedisplayed libraries, and competition studies indicated that many of the peptides bind at or near the carbohydrate binding site. Surprisingly, the peptides isolated by each mAb fell into distinct consensus-sequence groups that discriminated between the mAbs, and in general, the peptides bound only to the mAbs used for their isolation. Similar results were obtained with polyclonal antibodies directed against synthetic oligosaccharide fragments of the streptococcal cell-wall polysaccharide. Thus, the peptides appear to be specific for their isolating antibodies and are not recognized by the same mechanism as their carbohydrate counterparts.Carbohydrates (CHOs) have proven to be valuable tools in demonstrating immunologic mimicry. Anti-idiotypic antibodies (Abs) directed against the V domains of anti-CHO Abs can, in some instances, elicit CHO-binding Ab responses when used themselves as immunogens (e.g., refs. 1-4). This has been attributed to chemical similarity (known as the ''internal image'') between an anti-idiotypic Ab and the corresponding CHO antigen (1). Likewise, crossreactive peptides have been identified for several anti-CHO mAbs (4-7). In one case, the peptide was shown to elicit Abs having the same idiotype as the cognate, anti-CHO mAb (5), and in another to elicit a CHObinding response (4).The work described here addresses the molecular basis of crossreactivity between CHO and protein antigens with Abs. Our goal was to determine if the crossreactive peptides recognized by anti-CHO Abs would bind by the same mechanism as the corresponding epitope on the CHO target; if so, the basis of crossreactivity would be structural mimicry. We assembled a panel of five closely related mAbs against the cell-wall polysaccharide (CWPS) of group A Streptococcus (GAS) and showed by oligosaccharide mapping studies that they indeed bind a similar, if not identical, epitope. Each of four anti-GAS CWPS mAbs and three polyclonal Abs (PCAbs) against synthetic oligosaccharide fragments of the GAS CWPS isolated peptides bearing unique, chemically distinct consensus sequences. Moreover, representative peptides from each consensus group were functionally specific, because they usually bound only to their isolating Ab. Thus, these Abs were more restricted in their peptide reactivity than in their CHO recognition. We conclude that the predominating basis of peptide recognition by anti-CHO Abs differs between Abs, with true CHO mimics being relatively rare. We propose that the antigenic mimicry observed for CHO-crossreactive peptides is determined mainly by the ...

The successful application of a new resin consisting of beaded polyethylene glycol polyacrylamide copolymer 5 (PEGAl90o) as a solid support for the chemical-enzymatic synthesis of glycopeptides is reported; the resin is mechanically stable, yet highly swelling in both organic solvents and aqueous buffers.

The lytic transglycosylases cleave the bacterial cell wall heteropolymer peptidoglycan with the same specificity as the muramidases (lysozymes), between the N-acetylmuramic acid and N-acetylglucosamine residues, with the concomitant formation of a 1,6-anhydromuramoyl residue. The putative catalytic residue in the family 3 lytic transglycosylase from Pseudomonas aeruginosa, Glu162 as identified by sequence alignment to the homologous enzyme from Escherichia coli, was replaced with both Ala and Asp by site-directed mutagenesis. Neither mutant enzyme differed structurally from the wild-type enzyme, as judged by CD spectroscopy, but both were enzymatically inactive confirming the essential role of Glu162 in the mechanism of action of this lytic transglycosylase. The bhexosaminidase inhibitor NAG-thiazoline was shown to inhibit the activity of lytic transglycosylase activity, thus providing the first direct evidence that the formation of the 1,6-anhydromuramoyl residue may proceed through an oxazolinium ion intermediate involving anchimeric assistance. Using surface plasmon resonance and difference absorbance spectroscopy, K d values of 1.8 and 1.4 mM, respectively, were determined for NAG thiazoline, while its parent compound N-acetylglucosamine neither inhibited nor appeared to bind the lytic transglycosylase with any significant affinity.

Finger millet is an ancient African cereal crop, domesticated 7000 years ago in Ethiopia, reaching India at 3000 BC. Finger millet is reported to be resistant to various fungal pathogens including Fusarium sp. We hypothesized that finger millet may host beneficial endophytes (plant-colonizing microbes) that contribute to the antifungal activity. Here we report the first isolation of endophyte(s) from finger millet. Five distinct fungal species were isolated from roots and predicted taxonomically based on 18S rDNA sequencing. Extracts from three putative endophytes inhibited growth of F. graminearum and three other pathogenic Fusarium species. The most potent anti-Fusarium strain (WF4, predicted to be a Phoma sp.) was confirmed to behave as an endophyte using pathogenicity and confocal microscopy experiments. Bioassay-guided fractionation of the WF4 extract identified four anti-fungal compounds, viridicatol, tenuazonic acid, alternariol, and alternariol monomethyl ether. All the purified compounds caused dramatic breakage of F. graminearum hyphae in vitro. These compounds have not previously been reported to have anti-Fusarium activity. None of the compounds, except for tenuazonic acid, have previously been reported to be produced by Phoma. We conclude that the ancient, disease-tolerant crop, finger millet, is a novel source of endophytic anti-fungal natural products. This paper suggests the value of the crops grown by subsistence farmers as sources of endophytes and their natural products. Application of these natural chemicals to solve real world problems will require further validation.

Glycosylation of a disaccharide containing N-acetylglucosamine with rhamnosyl and mannosyl trichloracetimidates under triethysilyl triflate catalysis led to the competitive formation of glycosyl imidates. While the rhamnosyl imidate could be rearranged to the thermodynamically favored trisaccharide, the mannosyl analogue was resistant to rearrangement. Glycosylation with perbenzylated thiorhamnosides activated with methyl triflate (MeOTf) gave the trisaccharide as well as the methyl imidate trisaccharide. The less reactive alpha-thioethyl donor led to a higher relative amount of methyl imidate trisaccharide to trisaccharide than the more reactive beta-thioglycoside. When using a more reactive thioethyl fucoside only the trisaccharide was obtained. Interestingly, the acceptor treated with MeOTf gave the N-methyl imidate that could be easily rhamnosylated and subsequently converted to the N-acetamido trisaccharide. This strategy to glycosylate O-4 of N-acetylglucosamine is under further investigation. Alternatively, bis-N-acetylation of the glucosamine prevented the formation of imidates and allowed the efficient synthesis of two Lewis A trisaccharide analogues.