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Heavy Chain V Region Diversity in the Duck-Billed Platypus (Ornithorhynchus anatinus): Long and Highly Variable Complementarity-Determining Region 3 Compensates for Limited Germline Diversity¹

Jeannette Johansson^*, Maria Aveskogh^*, Barry Munday and Lars Hellman^2,*

^* Department of Cell and Molecular Biology, Uppsala University, Uppsala, Sweden; and Department of Human Life Sciences, University of Tasmania, Launceston, Tasmania, Australia

In this work, to study the emergence of the H chain V region repertoire during mammalian evolution, we present an analysis of 25 independent H chain V regions from a monotreme, the Australian duck-billed platypus, Ornithorhynchus anatinus. All the sequences analyzed were found to form a single branch within the clan III of mammalian V region sequences in a distance tree. However, compared with a classical V gene family this branch was more diversified in sequence. Sequence analysis indicates that the apparent lack of diversity in germline V segments is well compensated for by relatively long and highly diversified D and N nucleotides. In addition, extensive sequence variation was observed in the framework region 3. Furthermore, at least five and possibly seven different J segments seem to be actively used in recombination. Interestingly, internal cysteine bridges in the complementarity-determining region (CDR)3 loop, or between the CDR2 and CDR3 loops, are found in 36% of the platypus V_H sequences. Such cysteine bridges have also been observed in cow, camel, and shark. Internal cysteine bridges may play a role in stabilizing long and diversified CDR3 and thereby have a role in increasing the affinity of the Ab-Ag interaction.

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