A single-domain antibody fragment in complex with RNase A: non-canonical loop structures and nanomolar affinity using two CDR loops.

BACKGROUND: Camelid serum contains a large fraction of functional heavy-chain antibodies - homodimers of heavy chains without light chains. The variable domains of these heavy-chain antibodies (VHH) have a long complementarity determining region 3 (CDR3) loop that compensates for the absence of the antigen-binding loops of the variable light chains (VL). In the case of the VHH fragment cAb-Lys3, part of the 24 amino acid long CDR3 loop protrudes from the antigen-binding surface and inserts into the active-site cleft of its antigen, rendering cAb-Lys3 a competitive enzyme inhibitor. RESULTS: A dromedary VHH with specificity for bovine RNase A, cAb-RN05, has a short CDR3 loop of 12 amino acids and is not a competitive enzyme inhibitor. The structure of the cAb-RN05-RNase A complex has been solved at 2.8 A. The VHH scaffold architecture is close to that of a human VH (variable heavy chain). The structure of the antigen-binding hypervariable 1 loop (H1) of both cAb-RN05 and cAb-Lys3 differ from the known canonical structures; in addition these H1 loops resemble each other. The CDR3 provides an antigen-binding surface and shields the face of the domain that interacts with VL in conventional antibodies. CONCLUSIONS: VHHs adopt the common immunoglobulin fold of variable domains, but the antigen-binding loops deviate from the predicted canonical structure. We define a new canonical structure for the H1 loop of immunoglobulins, with cAb-RN05 and cAb-Lys3 as reference structures. This new loop structure might also occur in human or mouse VH domains. Surprisingly, only two loops are involved in antigen recognition; the CDR2 does not participate. Nevertheless, the antigen binding occurs with nanomolar affinities because of a preferential usage of mainchain atoms for antigen interaction.

Camel single-domain antibody inhibits enzyme by mimicking carbohydrate substrate.

Whereas antibodies have demonstrated the ability to mimic various compounds, classic heavy/light-chain antibodies may be limited in their applications. First, they tend not to bind enzyme active site clefts. Second, their size and complexity present problems in identifying key elements for binding and in using these elements to produce clinically valuable compounds. We have previously shown how cAb-Lys3, a single variable domain fragment derived from a lysozyme-specific camel antibody naturally lacking light chains, overcomes the first limitation to become the first antibody structure observed penetrating an enzyme active site. We now demonstrate how cAb-Lys3 mimics the oligosaccharide substrate functionally (inhibition constant for lysozyme, 50 nM) and structurally (lysozyme buried surface areas, hydrogen bond partners, and hydrophobic contacts are similar to those seen in sugar-complexed structures). Most striking is the mimicry by the antibody complementary determining region 3 (CDR3) loop, especially Ala104, which mimics the subsite C sugar 2-acetamido group; this group has previously been identified as a key feature in binding lysozyme. Comparative simplicity, high affinity and specificity, potential to reach and interact with active sites, and ability to mimic substrate suggest that camel heavy-chain antibodies present advantages over classic antibodies in the design, production, and application of clinically valuable compounds.

Comparison of llama VH sequences from conventional and heavy chain antibodies.

Forty different PCR clones encoding a llama variable heavy chain domain were analysed. The majority of these clones are derived from heavy-chain antibody cDNA in which the entire CH1 exon is absent. It appears from the amino acid within the VHH framework 1 and 3 that all the llama clones belong to the VH III family. However, the individual llama VHH sequences differ more substantially from each other than expected for members of the same family. Several remarkable amino acid substitutions in the framework 2 hinder the proper association of the VL. However, they lay the foundation for the secretion from the endoplasmic reticulum and good solubility behaviour of llama H2 antibodies. The repertoire of the llama VHHs may be extensive due to the presence of a long CDR3-loop, often constrained by a disulfide bridge and the occurrence of H1 and H2 loop conformations not yet encountered in mice or human VHs. The variability plot of the amino acids in the VHH shows that the first hypervariable region coincides with the structural H1 loop in contrast to the situation found in mice and man where the CDR1 and H1 are slightly offset. We propose that the amino acids of the llama H1 loop participate actively in the antigen binding. All these observations are characteristic for the llama VHHs of the homodimeric heavy-chain H2 antibodies, but are not maintained in the llama clones from conventional heterotetrameric H2L2 immunoglobulins.

Crystal structure of a camel single-domain VH antibody fragment in complex with lysozyme.

The Camelidae is the only taxonomic family known to possess functional heavy-chain antibodies, lacking light chains. We report here the 2.5 A resolution crystal structure of a camel VH in complex with its antigen, lysozyme. Compared to human and mouse VH domains, there are no major backbone rearrangements in the VH framework. However, the architecture of the region of VH that interacts with a VL in a conventional FV is different from any previously seen. Moreover, the CDR1 region, although in sequence homologous to human CDR1, deviates fundamentally from the canonical structure. Additionally, one half of the CDR3 contacts the VH region which in conventional immunoglobulins interacts with a VL whereas the other half protrudes from the antigen binding site and penetrates deeply into the active site of lysozyme.

Frequency and DNA content of micronuclei in rat parenchymal liver cells during experimental hepatocarcinogenesis.

Liver carcinogenesis is considered to be a good experimental model to study the sequential changes leading to cancer and was applied here for the analysis of chromosome/genome mutations. Since the micronucleus test was shown to be an adequate method to detect and analyse chromosome changes in dividing cells, the frequency of micronuclei (MN) together with their relative DNA content (DNA content of the MN divided by the DNA content of the corresponding nucleus) were analysed in hepatocytes isolated from rats at different stages of experimentally induced hepatocarcinogenesis. The protocol used for the induction of liver cancer was based on the triphasic 'Gerlans protocol', a Solt-Farber procedure supplemented with a phenobarbital (PB) promotion step. Male Wistar rats were initiated by a single i.p. dose of diethylnitrosamine (DENA), followed by selection of the resistant hepatocytes by 2-acetylaminofluorene (2-AAF). Subsequent promotion was accomplished by chronic exposure to phenobarbital. For each group of rats a mitotic stimulator (CCl4) is necessary at the end of their treatment period to express the clastogenic and/or aneugenic lesions which may lead to micronuclei. The results of these experiments do confirm that genetic alterations are occurring at the chromosome level (MN expression) during the different steps of experimental rat liver carcinogenesis. DNA measurements seem to be a good genetic parameter to detect eventual differences between the chromosomal content (whole chromosome or chromosome fragments) of MN populations appearing in different stages of the carcinogenic process. Moreover, a comparison between the mono- and bi-nucleated cell population showed that the frequency of micronuclei is higher in mononuclear parenchymal liver cells.