Stephen B. H. Kent

A cellular gene encodes scrapie PrP 27-30 protein.

A clone encoding PrP 27-30, the major protein in purified preparations of scrapie agent, was selected from a scrapie-infected hamster brain cDNA library by oligonucleotide probes corresponding to the N terminus of the protein. Southern blotting with PrP cDNA revealed a single gene with the same restriction patterns in normal and scrapie-infected brain DNA. A single PrP-related gene was also detected in murine and human DNA. PrP-related mRNA was found at similar levels in normal and scrapie-infected hamster brain, as well as in many other normal tissues. Using antisera against PrP 27-30, a PrP-related protein was detected in crude extracts of infected brain and to a lesser extent in extracts of normal brain. Proteinase K digestion yielded PrP 27-30 in infected brain extract, but completely degraded the PrP-related protein in normal brain extract. No PrP-related nucleic acids were found in purified preparations of scrapie prions, indicating that PrP 27-30 is not encoded by a nucleic acid carried within the infectious particles.

Identification and Chemical Synthesis of a Host Cell Receptor Binding Site on Hepatitis B Virus

Hepatitis B virus (HBV) has not yet been propagated in vitro, and knowledge concerning its reaction with receptors on target cells remained scant. We have located within the HBV envelope proteins a sequence mediating the attachment of HBV to human hepatoma HepG2 cells. A synthetic peptide analog (PLGFFPDHQLDPAFGANSNNPDWDFNP) is recognized by both cell receptors and anti-HBV antibodies and elicits antibodies reacting with native HBV. The synthetic peptide is a promising immunogen expected to elicit protective antibodies based on the concept of the attachment blockade pathway of virus neutralization. The approach described here, based on anti-peptide antisera and the binding of peptide analogs to cell receptors is generally applicable for the delineation of cell receptor binding sites on viruses with known gene sequences.

Purification and structural studies of a major scrapie prion protein

Scrapie is a degenerative, neurological disorder caused by a slow infectious agent or prion. Extensively purified preparations of prions were denatured by boiling in sodium dodecyl sulfate and the major protein component (PrP 27-30) was isolated by preparative HPLC size exclusion chromatography after proteinase K digestion. The purified PrP 27-30 molecules were not infectious. Ultraviolet absorption spectra of purified PrP 27-30 demonstrated the absence of covalently linked polynucleotides. Amino acid composition studies showed that PrP 27-30 contains at least 17 naturally occurring amino acids. A single N-terminal amino acid sequence for PrP 27-30 was obtained; the sequence is N-Gly-Gln-Gly-Gly-Gly-Thr-His-Asn-Gln-Trp-Asn-Lys-Pro-Ser-Lys and it does not share homology with any known proteins. The same amino acid sequence was found when an extensively purified preparation of prions aggregated into rods and containing approximately 10(9.5) ID50 U/ml was sequenced directly. Knowledge of the amino acid sequence should permit determination of the genetic origin and replication mechanism of prions.

Enzymatic activity of a synthetic 99 residue protein corresponding to the putative HIV-1 protease

A protein corresponding to the putative protease of the human immunodeficiency virus 1 (HIV-1) has been prepared by total chemical synthesis. This 99 residue synthetic enzyme showed specific proteolytic activity on fragments of the natural gag precursor and on synthetic peptide substrates, two of which released fragments corresponding to the N terminus and C terminus of the protease molecule itself. The observed substrate specificity was not restricted to cleavage at Phe/Tyr-Pro bonds. Inhibition studies provided direct evidence that the HIV-1 protease belongs to the family of aspartic proteases. The availability of the HIV-1 protease as a defined molecular species has important implications for the design of specific inhibitors that do not interfere with the host cell metabolism as a possible route to antiviral agents against acquired immunodeficiency syndrome (AIDS).

Antibodies to a synthetic peptide from the preS 120–145 region of the hepatitis B virus envelope are virus-neutralizing

Studies with synthetic peptides have provided evidence for the presence of preS coded sequences in the envelope (env) proteins of hepatitis B virus (HBV) and indicated that these sequences are involved in the specific attachment of HBV to liver cells. Scanning of the preS sequence for immunodominant continuous epitopes identifies the sequence within residues preS (120-145) as the most immunogenic in eliciting antibodies recognizing HBV and as the most efficiently binding antibodies from sera of rabbits and humans immunized with HBV env proteins. To assess the potential of preS (120-145) as a synthetic vaccine against hepatitis B, in vitro neutralization of the virus by rabbit antiserum to the peptide was assayed in chimpanzees. The animals, subsequently proven to be susceptible to HBV infection, did not develop hepatitis B as judged by negative serological tests for HBV-associated antigens and antibodies and by normal serum alanine aminotransferase levels and normal liver biopsies. These results establish the role of preS domains in the process of virus neutralization and the potential of synthetic preS analogues for hepatitis B vaccination.

Antibodies to Hepatitis B Surface Antigen (HBsAg) Elicited by Immunization with a Synthetic Peptide Covalently Linked to Liposomes

Peptides synthesized for potential application as antiviral vaccines have been mostly tested in the form of conjugates with carrier proteins. The possible use of several distinct synthetic vaccines in prophylaxis would be facilitated by the availability of fully synthetic immunogens. A synthetic peptide corresponding to residues 135 to 155 ( P135 -155) of hepatitis B surface antigen (HBsAg) failed to elicit in free form anti-peptide antibodies or anti-HBs. However, polymers of P135 -155 (prepared by linking to diaminoalkanes ) and synthetic conjugates prepared by binding P135 -155 to liposomes or polylysine were immunogenic. A poor correlation was observed between anti-peptide and anti-HBs responses elicited by these conjugates. Glutaraldehyde-fixed liposomes appeared to be the carriers of choice for inducing anti-HBs.

Delineation of contiguous determinants essential for biological functions of the pre-S sequence of the hepatitis B virus envelope protein: its antigenicity, immunogenicity and cell-receptor recognition

The preS region of the hepatitis B virus (HBV) envelope protein has the following properties: (1) exposure on the surface of the virus; (2) high immunogenicity; (3) involvement in the reaction of the virus with cell receptors and (4) elicitation of antibodies protective against infection. Attempts to mimic B- and T-cell epitopes on the native protein by synthetic peptides were highly successful. This success depended on identification of those regions within the preS sequences which are the most important for biological function of the virus and for immunity, and on the synthesis of long peptides (20-40 residues) containing both B- and T-cell epitopes. Results presented here highlight those subregions of the preS sequence which are the most essential for the antigenicity and immunogenicity of HBV.

Detection of Antiviral Antibodies with Predetermined Specificity Using Synthetic Peptide-beta-Lactamase Conjugates: Application to Antibodies Specific for the preS Region of the Hepatitis B Virus Envelope Proteins

Amino acid sequences coded for by the preS region of the hepatitis B virus (HBV) envelope gene are present both in HBV and in subviral hepatitis B surface antigen (HBsAg) particles. Consequently, anti-preS-specific antibodies are elicited during the course of HBV infection. Such antibodies are virus-neutralizing. Therefore, it is important to determine whether or not vaccination with HBsAg also induces an anti-preS-specific immune response. We describe here an enzyme-linked immunosorbent assay applicable for the screening of sera from vaccinated individuals for anti-preS antibodies. IgG from serum specimens was adsorbed to staphylococcal Protein A on a superparamagnetic support and subsequently mixed with a synthetic peptide analogue [preS(120-145)] covalently linked to beta-lactamase. The presence of anti-preS in serum specimens resulted in binding of the conjugated beta-lactamase to the magnetic support. The adsorbed enzyme was quantified colorimetrically.

Protein microsequencing with post-column fluorescent phenylisothiocyanate analogues

Abstract A novel class of isothiocyanates has been introduced for protein microsequencing. These modified phenylisothiocyanates (PITC) are substituted in the 4-position of the phenyl ring with a protected amine and have a general structure tert. -Boc-NH-(CH 2 ) n ,-PITC. The most important feature of these compounds is that a primary amine is generated during the acid cleavage step of the sequencing process. This primary amine is then available for fluorescent labeling at the time of identification to increase the detectibility of the modified phenylthiohydantoin (PTH)-amino acid. Preliminary studies were performed on aniline, benzylamine and phenethylamine to evaluate the optimal mode for fluorescent post-column detection. Following synthesis of 4-(Boc-amino)PITC and 4-(Boc-aminomethyl)PITC, these compounds were found to sequence polypeptides with repetitive yields similar to PITC. Preliminary studies on the fluorescence detection of the aminomethylPTH-amino acids showed at least a 2.5-fold increase in sensitivity over the most commonly used detection method, UV detection at 254 nm. These results indicate that this approach may provide a general method for picomole and subpiconole protein sequence analysis using currently available instrumentation.

Microscale structure analysis of a high-molecular-weight, hydrophobic membrane glycoprotein fraction with platelet-derived growth factor-dependent kinase activity

General methods for the study of the primary structure of picomole quantities of large, hydrophobic membrane glycoproteins with blocked amino-termini have been developed. Three techniques designed to be used in concert with each other are described: first, modified protein preparation and fragmentation techniques; secondly, a simple but very selective two-dimensional reversed-phase high-performance liquid chromatography system for the resolution of complex mixtures of small to medium-sized tryptic peptides on Vydac C4, C18 and diphenyl columns and thirdly, a two-dimensional separation method for large, denaturated (CNBr) polypeptide fragments by size-exclusion high-performance liquid chromatography, combined with either reversed-phase high-performance liquid chromatography (C4) or sodium dodecyl sulphate polyacrylamide gel electrophoresis in conjunction with electroblotting and autoradiography. These methods were applied to studies of the platelet-derived growth factor receptor. Starting with 500 pmoles of purified protein, a total of 232 amino acids were sequenced.