Investigation of factors influencing the immunogenicity of hCG as a potential cancer vaccine.

Human chorionic gonadotrophin (hCG) and its ß-subunit (hCGß) are tumour autocrine growth factors whose presence in the serum of cancer patients has been linked to poorer prognosis. Previous studies have shown that vaccines which target these molecules and/or the 37 amino acid C-terminal hCGß peptide (hCGßCTP) induce antibody responses in a majority of human recipients. Here we explored whether the immunogenicity of vaccines containing an hCGß mutant (hCGßR68E, designed to eliminate cross-reactivity with luteinizing hormone) or hCGßCTP could be enhanced by coupling the immunogen to different carriers [keyhole limpet haemocyanin (KLH) or heat shock protein 70 (Hsp70)] using different cross-linkers [1-ethyl-3(3-dimethylaminopropyl)carboiimide (EDC) or glutaraldehyde (GAD)] and formulated with different adjuvants (RIBI or Montanide ISA720). While there was little to choose between KLH and Hsp70 as carriers, their influence on the effectiveness of a vaccine containing the BAChCGßR68E mutant was less marked, presumably because, being a foreign species, this mutant protein itself might provide T helper epitopes. The mutant provided a significantly better vaccine than the hCGßCTP peptide irrespective of the carrier used, how it was cross-linked to the carrier or which adjuvant was used when hCG was the target. Nonetheless, for use in humans where hCG is a tolerated self-protein, the need for a carrier is of fundamental importance. Highest antibody titres were obtained by linking the BAChCGßR68E to Hsp70 as a carrier by GAD and using RIBI as the adjuvant, which also resulted in antibodies with significantly higher affinity than those elicited by hCGßCTP peptide vaccine. This makes this mutant vaccine a promising candidate for therapeutic studies in hCGß-positive cancer patients.

Inhibition of human beta-tryptase by Bowman-Birk inhibitor derived peptides.

Four 11-residue peptides based on the Bowman-Birk inhibitor (BBI) structure were synthesized. These were tested for their ability to inhibit human beta-tryptase. Peptides with a basic residue at P1 inhibited tryptase even though the intact BBI protein is inactive. This result is interpreted in terms of the unique structural arrangement of active sites in tryptase which prevent access by large protein inhibitors.

Synthetic peptide mimics of the Bowman-Birk inhibitor protein.

Proteins of the Bowman-Birk inhibitor family of serine proteinase inhibitors interact with the enzymes they inhibit via an exposed surface loop that adopts the canonical proteinase inhibitory conformation. The resulting non-covalent complex renders the proteinase inactive. This inhibition mechanism is common for the majority of serine proteinase inhibitor proteins and many analogous examples are known. A particular feature of the Bowman-Birk inhibitor protein, however, is that the interacting loop is a particularly well-defined disulfide-linked short beta-sheet region. Moreover, synthetic peptides based on this region keep the same structure as the corresponding part of the full sized protein and also retain inhibitory activity. This review describes the background to inhibition by Bowman-Birk inhibitor proteins (and derived peptides) and shows how peptides based on the reactive site can be manipulated in order to generate potent proteinase inhibitors with redirected specificities.

The Bowman-Birk inhibitor reactive site loop sequence represents an independent structural beta-hairpin motif.

We have determined the NMR structure in aqueous solution of a disulphide-cyclised 11-residue peptide that forms a stable beta-hairpin, incorporating a type VIb beta-turn. The structure is found to be extremely well ordered for a short peptide, with the 30 lowest energy simulated annealing structures having an average pairwise r.m.s. deviation of only 0.36 A over the backbone. All but three side-chains adopt distinct conformations, allowing a detailed analysis of their involvement in cross-strand interactions. The peptide sequence analysed originates from a previously reported study, which identified potent inhibitors of human leukocyte elastase from screening a combinatorial peptide library based on the short protein beta-sheet segment that forms the reactive site loop of Bowman-Birk inhibitors. A detailed comparison of the peptide's solution structure with the corresponding region in the whole protein structure reveals a very good correspondence not only for the backbone (r.m.s. deviation approximately 0.7 A) but also for the side-chains. This isolated beta-hairpin retains the biologically active "canonical conformation" typical of small serine proteinase inhibitor proteins, which explains why it retains inhibitory activity. Since the structural integrity is sequence-inherent and does not depend upon the presence of the remaining protein, this beta-hairpin represents an independent structural motif and so provides a useful model of this type of protein architecture and its relation to biological function. The relationship between the conformation of this beta-hairpin and its biological activity is discussed.

Identification of chymotrypsin inhibitors from a second-generation template assisted combinatorial peptide library.

In an earlier study (McBride JD, Freeman N, Domingo GJ, Leatherbarrow RJ. Selection of chymotrypsin inhibitors from a conformationally-constrained combinatorial peptide library. J. Mol. Biol. 1996; 259: 819-827) we described a resin-bound cyclic peptide library, constructed based on the sequence of the anti-tryptic reactive site loop of Bowman Birk Inhibitor (BBI), a proteinase inhibitor protein. This library was used to identify re-directed chymotrypsin inhibitors with Ki values as low as 17 nM. We have now extended this work by constructing an enhanced library in which a further position, at the P4 site of the inhibitor, has been randomized. This new library has variation at three target locations (P4, P1 and P2) within the inhibitory loop region, producing 8,000 variants. Screening this library allowed selection of new inhibitor sequences with Ki values as low as 3.4 nM. The success of this approach is reflected by the fact that the inhibition constant given by the selected peptide sequence is slightly lower than that reported against chymotrypsin for the most studied full length BBI protein, Soybean BBI 2-IV.

Selection of human elastase inhibitors from a conformationally constrained combinatorial peptide library.

A resin-bound cyclic peptide library was constructed based on the sequence of the reactive-site loop of Bowman-Birk inhibitor, a proteinase inhibitor protein. The constrained loop sequence, which incorporates the minimal proteinase-binding motif, was retained throughout the library, but selected residues known to be important for inhibitor specificity were randomised. The approach was used to create a 'one bead, one peptide' library with 8000 variants resulting from randomization at three target locations in the sequence (P4, P1 and P2'). This library allows us to examine the degree to which variations in this proteinase-binding motif can redirect activity, as well as providing information about the binding specificity of a proteinase target. Screening this library for binding to human leucocyte elastase identified sequences with a strong consensus, and on resynthesis all were found to act as inhibitors, with Ki values as low as 65 nM. Human leucocyte elastase is known to have a substrate preference for small alkyl chains at the P1 locus, with valine being preferred. However, alanine and not the expected valine was found in 21 out of 23 identified sequences. The remaining two sequences had threonine at P1, a finding that would be hard to predict based on substrate specificity alone. Further analysis of resynthesized peptides demonstrated that valine substitution results in an analogue that is hydrolysed far more rapidly than ones having library-selected P1 residues. Testing of the human leucocyte elastase-selected sequences as inhibitors of porcine pancreatic elastase demonstrates a significant difference in the specificity of the P4 locus between these two proteinases.

The role of the P2' position of Bowman-Birk proteinase inhibitor in the inhibition of trypsin. Studies on P2' variation in cyclic peptides encompassing the reactive site loop.

The role of the P2' residue in proteinase inhibitors of the Bowman-Birk family was investigated using synthetic cyclic peptides based on the reactive site loop of the inhibitor. A series of 21 variants having different P2' residues was tested for inhibition of trypsin, and the rate at which they were hydrolysed by this enzyme was also measured. Variation at P2' was found to result in marked differences in inhibitory potency, with the best sequence (Ile) having a Ki value of 9 nM. Peptides with P2' Gly, Pro or Glu failed to demonstrate any measurable inhibition (Ki>1 mM). The peptides also displayed significant differences in the rates at which they were hydrolysed, which varied by over three orders of magnitude between the difference sequences. There was found to be overall correlation between the Ki value and the rate of hydrolysis, with peptides that inhibited best also being hydrolysed more slowly. The results are discussed in light of the sequence information for Bowman-Birk inhibitor proteins.

The role of threonine in the P2 position of Bowman-Birk proteinase inhibitors: studies on P2 variation in cyclic peptides encompassing the reactive site loop.

Previously, we have described a template-assisted combinatorial peptide library based on the anti-tryptic reactive site loop of a Bowman-Birk inhibitor (BBI). Sequences that displayed inhibitory activity re-directed towards chymotrypsin were found to have a consensus binding motif, with their most striking feature being that exclusively threonine was found at the P2 position. The present study investigates the reason for this surprising specificity by maintaining the binding motif but systematically varying the P2 residue. From analysis of 26 variants, it is found that the requirements for inhibitory activity at P2 are finely tuned, and in agreement with the library work, threonine at P2 provides optimal inhibition. In addition, peptides with threonine at P2 are significantly less susceptible to hydrolysis. Examination of all available BBI sequences shows that threonine is very highly conserved at P2, which implies that the functional requirement extends to the full-length BBI protein. Our results are consistent with a dual requirement for hydrophobic recognition within the S2 pocket and maintenance of an inhibitory conformation via hydrogen bonding within the reactive-site loop. As the isolated peptide loop reproduces the active region of full-length BBI, these results explain why threonine is well conserved at P2 in this class of inhibitor. Furthermore, they illustrate that proteinase inhibitor specificity can have characteristics that are not easily predicted from information on the substrate preferences of a proteinase.

Selection of chymotrypsin inhibitors from a conformationally-constrained combinatorial peptide library.

A synthetic library of cyclic peptides was constructed utilizing the anti-tryptic loop region of the Bowman-Birk inhibitor, D4 from Macrotyloma axillare, as a template. The loop region of this proteinase inhibitor was reproduced by an 11 residue sequence, conformationally constrained by the presence of a disulfide bridge, to act as a mimetic of the functional reactive site region of this protein. This sequence, plus a pentaglycine spacer arm, was used to create a "one bead, one peptide" combinatorial library after on-resin deprotection and cyclization. Randomization at three positions considered to be important for proteinase specificity (P2, P1 and P'2) with the genetically coded amino acids (minus cysteine) plus norleucine generated 8000 permutations. Screening this library with biotinylated alpha-chymotrypsin under appropriate conditions revealed a small number (<0.05%) of beads that selectively bound the labeled proteinase. The sequences present on these active beads were determined, and found to have a well-defined consensus. Analysis of chymotrypsin inhibition in solution using re-synthesized peptides reveals that the sequences identified are potent inhibitors with Ki values in the nanomolar range. These results show that directed randomization of the canonical loop is a powerful way of generating proteinase inhibitors with targeted specificities. Incorporation of selective random changes within a defined structural framework is found to be an effective means of generating variation in large synthetic systems. The functional basis for inhibition by the identified sequences is discussed.

Electrophoretic detection of chitinase isoenzymes using the PhastSystem.

Zymographic assays are described for the detection of chitinase isoenzymes following isoelectric focusing. Method 1 used a polyacrylamide overlay gel containing glycol chitin. Following hydrolysis, visualisation of isoenzymes was achieved by fluorescent counter-staining of the gel with Fluorescent Brightener 28. Method 2 used a cellulose acetate membrane overlay which incorporates 4-methylumbelliferyl substrates, hydrolysis of which released a fluorescent product (4-methylumbelliferone). The pIs of chitinase isoenzymes were estimated by using coloured pI markers. A significant time advantage was obtained over previous methods. The method was used to demonstrate chitinase activity in control rat lungs and in rat lungs infected with the pathogen Pneumocystis carinii.

Alterations in cysteine proteinase content of rat lung associated with development of Pneumocystis carinii infection.

The rate of hydrolysis of three cysteine-type proteinase substrates, N-benzyloxycarbonyl-Arg-Arg-4-methyl-7-coumarylamide (AMC) (cathepsin B), Arg-AMC (cathepsin H), and N-benzyloxycarbonyl-Phe-Arg-AMC (cathepsin L), were determined in rat lung throughout the time course of the induction of Pneumocystis carinii infection by immunosuppression. Cathepsin B-like and cathepsin L-like activities fell below control values initially, but from week 8 of the immunosuppressive treatment significant increases above the control were noted. Cathepsin H-like activity was greater than control levels from week 3, and by week 12 it was 7,600% of the mean control value. When compared with the relative degree of infection, as assessed from the number of cysts present in lung impression smears, cathepsin B-like and cathepsin L-like activities were significantly increased only at heavy parasite burdens while cathepsin H-like activity displayed a close correlation with parasite number (r = 0.884; P less than 0.001). Activity was detected in lysates of purified P. carinii with all three substrates. Treatment of heavily infected animals with co-trimoxazole cleared the lungs of P. carinii, and this was accompanied by a marked reduction in proteinase activity, in particular, cathepsin H-like activity, which fell from 108- to 3-fold the mean control value following drug treatment. Analysis of cathepsin H isozyme patterns by fluorography following isoelectric focusing revealed differences between treated and control lung samples. In the immunosuppressed group, there was a time-dependent increase in the intensity of some of the bands observed in the controls and an appearance of several novel bands which corresponded to bands observed in lysates of P. carinii. It is likely, therefore, that the increased proteinase activity observed in the treated group is due, at least in part, to isozymes from P. carinii; consequently, cathepsin H-like activity might be of use diagnostically in the identification of P. carinii infection and in the estimation of parasite burden.