Identification and characterization of a peptide that specifically binds the human, broadly neutralizing anti-human immunodeficiency virus type 1 antibody b12.

Human monoclonal antibody (MAb) b12 recognizes a conformational epitope that overlaps the CD-4-binding site of the human immunodeficiency virus type 1 (HIV-1) envelope. MAb b12 neutralizes a broad range of HIV-1 primary isolates and protects against primary virus challenge in animal models. We report here the discovery and characterization of B2.1, a peptide that binds specifically to MAb b12. B2.1 was selected from a phage-displayed peptide library by using immunoglobulin G1 b12 as the selecting agent. The peptide is a homodimer whose activity depends on an intact disulfide bridge joining its polypeptide chains. Competition studies with gp120 indicate that B2.1 occupies the b12 antigen-binding site. The affinity of b12 for B2.1 depends on the form in which the peptide is presented; b12 binds best to the homodimer as a recombinant polypeptide fused to the phage coat. Originally, b12 was isolated from a phage-displayed Fab library constructed from the bone marrow of an HIV-1-infected donor. The B2.1 peptide is highly specific for b12 since it selected only phage bearing b12 Fab from this large and diverse antibody library.

The maltose-binding protein as a scaffold for monovalent display of peptides derived from phage libraries.

Random peptide libraries are displayed on filamentous bacteriophage as fusions to either the minor coat protein, pIII, or the major coat protein, pVIII. We have devised a means of isolating the peptide displayed on a phage clone by transferring it to the N-terminus of the maltose-binding protein (MBP) of Escherichia coli encoded by malE. Transfer of a peptide sequence to monomeric MBP eliminates phage-encoded amino acids downstream of the insert peptide as well as avidity effects caused by multivalent display on phage. Peptide:MBP fusions are also easily affinity purified on amylose columns. The pMal-p2 vector was engineered to accept phage DNA encoding pIII- and pVIII-displayed peptides fused to their respective leader sequences. Both types of leader sequence were shown to target the peptide:MBP fusions to the periplasm of E. coli. A streamlined procedure for transferring peptides to MBP was applied to clones that had been isolated from a panel of pVIII-displayed peptide libraries by screening with an HIV-1-specific monoclonal antibody (Ab). By enzyme-linked immunosorbent assay, the Ab bound each of the peptide:MBP fusions and required the presence of a disulfide bridge within each peptide. Some of the peptide:MBP fusions were also analyzed using surface plasmon resonance. Thus, our study shows the value of malE fusion vectors in characterizing phage-displayed peptides.

Epitope mapping of a function-blocking beta 1 integrin antibody by phage display.

Integrins are a major class of cell surface receptors involved in cell-cell and cell-matrix adhesion and communication. Ha2/11 is a function-blocking anti-rat beta 1 integrin hamster IgM that should be a useful reagent for understanding beta 1 integrin function. We demonstrate that Ha2/11 cross reacts with human, Xenopus, and Drosophila beta 1 integrins, and use phage display to map the epitope for Ha2/11 to residues within the sequence LRSGEPQTF which lies 18 amino acids proximal to the putative I domain in beta 1 integrins. Monoclonal antibody mapping experiments, mutational analyses, and direct binding assays have implicated integrin I domains in both cation and ligand binding. Our data therefore suggest that Ha2/11 blocks beta 1 integrin function by interfering with I domain-mediated ligand binding.

An expression map from human chromosome 14q24.3.

We have constructed an expression map of chromosome 14q24.3 between markers D14S42 and D14S63. cDNA selection with YACs from 14q24.3 was used to generate expressed sequence tags (ESTs). The localization of ESTs was confirmed on a YAC contig. PCR products of ESTs were used as probes to screen cDNA libraries leading to the isolation of transcripts for known and unknown genes. In total, the expression map contains 7 known genes previously mapped to 14q24.3, 6 cDNA transcripts, and 15 anonymous ESTs. The addition of 21 unique transcribed loci from an approximately 5- to 7-Mb region of chromosome 14q24.3 will facilitate future efforts to identify human disease genes from this region.

Assaying phage-borne peptides by phage capture on fibrinogen or streptavidin.

There is no simple and efficient method for assaying phage isolated from libraries without having to resort to PEG purification of the phage, or to the biotinylation or other labelling of the target molecule. We report here a method for producing 'bifunctional' phage that express two types of peptide; one peptide, fused to pVIII, will bind to immobilized fibrinogen, allowing capture of the phage out of culture supernatants; this allows the other peptide, fused to pIII or pVIII to be assayed by simple ELISA. This system has also been developed for the capture of phage bearing a streptavidin-binding peptide. The bifunctional phage are produced by bacterial cells bearing a plasmid that expresses pVIII fused either to the fibrinogen-binding peptide or to the streptavidin-binding one. Thus, when these cells are infected with a phage clone or pool to be assayed, phage will be produced whose 'capture-peptide' is produced from the plasmid and whose 'assay-peptide' is produced from the phage genome. We show here that, by this method, bifunctional phage can be produced that will bind to immobilized streptavidin or fibrinogen.

Probing the basis of antibody reactivity with a panel of constrained peptide libraries displayed by filamentous phage.

The structural requirements for peptide binding to an antibody may be elucidated by probing it with a variety of peptides having different constraints. To this end, we have constructed and screened a panel of peptide libraries displayed by filamentous bacteriophage. The peptides in most of the libraries have the potential for constraint by fixed Cys residues, which have been placed at different sites within a randomized amino acid sequence of varying length. When taken together, the binding data obtained from screening the panel with a given antibody allow one to determine the types of constraints that promote binding, as well as the residues that are critical for binding. We describe the construction of 11, pVIII-displayed, peptide libraries, whose sizes range from 150 million to 10 billion clones. The libraries were screened with a number of polyclonal and monoclonal antibodies against peptides, proteins and carbohydrates. Cross-reactivity with peptides was always found for antibodies produced against peptides, linear epitopes on folded proteins and, surprisingly, carbohydrates, whereas antibodies against discontinuous epitopes on proteins were found less frequently. The implications of these results are discussed in terms of the structural basis for cross-reactivity with peptides.

Cloning, sequencing, and mapping of the human chromosome 14 heat shock protein gene (HSPA2).

A genomic clone for the human heat shock protein (HSP) 70 gene located on chromosome 14 was isolated and sequenced. The gene, designated HSPA2, has a single open reading frame of 1917 bp that encodes a 639-amino acid protein with a predicted molecular weight of 70,030 Da. Analysis of the sequence indicates that HSPA2 is the human homologue of the murine Hsp70-2 gene with 91.7% identity in the nucleotide coding sequence and 98.2% in the corresponding amino acid sequence. HSPA2 has less amino acid homology to other members of the human HSP70 gene family, 83.3% to the heat-inducible HSP70-1 gene and 86.1% with the human heat shock cognate gene HSC70. HSPA2 is constitutively expressed in most tissues, with very high levels in testis and skeletal muscle. Significant but lower levels are also expressed in ovary, small intestine, colon, brain, placenta, and kidney. A yeast artificial chromosome (YAC) clone containing HSPA2 (YAC741H4) that also contained the polymorphic marker D14S63 was identified. This 670-kb YAC was mapped to 14q24.1 by fluorescence in situ hybridization (FISH). Subsequent two-color FISH and genetic mapping placed HSPA2/D14S63 proximal to the markers D14S57 and D14S77.

The c-fos gene and early-onset familial Alzheimer's disease.

A gene for early-onset familial Alzheimer's disease (FAD) is located on chromosome 14q24.3. The c-fos gene (FOS) is also located in the same band of this chromosome and is thus a candidate for the FAD locus. A yeast artificial chromosome (YAC) clone was identified which contains FOS. This YAC also contains the short-tandem repeat polymorphic (STRP) locus D14S76, placing FOS in the FAD region between D14S53 and D14S43. No recombinants were observed between D14S76 and FAD, and a maximum positive LOD score of 7.20 at a recombination fraction of 0.001 was observed for linkage of this marker to FAD. DNA sequence analysis was used to examine FOS in two affected subjects from an FAD family in which the chromosome 14 FAD locus is clearly responsible for the disease. The coding regions and parts of the 5' and 3' untranslated sequences of FOS were sequenced; no FAD-related mutations were observed. This work suggests that the FOS gene is not the chromosome 14 FAD locus although we cannot exclude the possibility that a mutation in an as yet unknown regulatory region is responsible for the disease. A new polymorphism was detected in the third intron of the gene.

Genetic linkage evidence for a familial Alzheimer's disease locus on chromosome 14.

Linkage analysis was used to search the genome for chromosomal regions harboring familial Alzheimer's disease genes. Markers on chromosome 14 gave highly significant positive lod scores in early-onset non-Volga German kindreds; a Zmax of 9.15 (theta = 0.01) was obtained with the marker D14S43 at 14q24.3. One early-onset family yielded a lod score of 4.89 (theta = 0.0). When no assumptions were made about age-dependent penetrance, significant results were still obtained (Zmax = 5.94, theta = 0.0), despite the loss of power to detect linkage under these conditions. Results for the Volga German families were either negative or nonsignificant for markers in this region. Thus, evidence indicates a familial Alzheimer's disease locus on chromosome 14.