Rapid Evolution of the Trophoblast Kunitz Domain Proteins (TKDPs)—A Multigene Family in Ruminant Ungulates Anindita Chakrabarty, 1 James A. MacLean II, 2 * Austin L. Hughes, 3 R. Michael Roberts, 1,2,4 Jonathan A. Green 4 1 Department of Veterinary Pathobiology, University of Missouri—Columbia, Columbia, Missouri 65211, USA 2 Department of Biochemistry, University of Missouri–Columbia, Columbia, Missouri 65211, USA 3 Department of Biological Sciences, University of South Carolina, Columbia, South Carolina 29208, USA 4 Department of Animal Sciences, University of Missouri—Columbia, Columbia, Missouri 65211, USA Received: 7 November 2005 / Accepted: 19 April 2006 [ReviewingEditor: Dr. Martin Kreitman] Abstract. The trophoblast Kunitz domain proteins (TKDPs) are products of the outer cells (tropho- blasts) of the placenta of cattle, sheep, and related species. Most are expressed abundantly for only a few days during the time at which the ruminant conceptus is first establishing intimate contacts with the uterine lining. The TKDPs are secretory proteins that possess a carboxyl-terminal peptidase inhibitory domain re- lated to the Kunitz family of serine peptidase inhib- itors. On the amino-terminal end are one or more highly unusual regions that are unique to the TKDP genes and have no apparent similarity to any other known sequences. The TKDPs are a rather divergent family that exhibits a good deal of variation among the members. To better understand the reason for such variation, the rates of synonymous (dS) and nonsynonymous (dN), as well as radical (p NR ) and conservative (p NC ), substitutions were assessed. Phy- logenetic trees revealed that the Kunitz domains represented three related groups, whereas the amino- terminal domains formed four groupings. Pairwise comparisons between Kunitz and amino-terminal domain groups demonstrated that dN was consis- tently greater than dS. In addition, nonsynonymous substitutions in the Kunitz domains tended to be radical (changing charge or polarity), while those in the amino-terminal domains exhibited neither a pre- ponderance of conservative nor radical substitution rates. In summary, the rapid evolution of the TKDPs, coupled with their restricted temporal expression during development, likely reflects the establishment of protein-protein interactions that have evolved to serve the unusual synepitheliochorial placenta of ruminant ungulates. Key words: Peptidase inhibitors — Placenta — Reproduction — Kunitz — Cattle — Sheep Introduction The trophoblast Kunitz domain proteins (TKDPs) are a multigene family expressed in the placenta of ruminant ungulates. To date, 10 have been cloned and described (5 from cattle and 5 from sheep) (MacLean et al. 2003). Each member of this family is charac- terized by a carboxyl-terminal Kunitz-type serine peptidase inhibitor domain. Kunitz domains are small globular motifs of 60 amino acids and most are held in a rigid conformation by the presence of three con- served disulfide bonds (Creighton and Charles 1987; Roberts et al. 1995). A loop projects from the core to interactwiththetargetedpeptidase.Withintheloopis *Current address: Department of Immunology, The University of Texas, M. D. Anderson Cancer Center, Houston, Texas 77054, USA Correspondence to: Jonathan A. Green, 163 ASRC, 920 East Campus Drive, University of Missouri—Columbia, Missouri 65211, USA; email: greenjo@missouri.edu J Mol Evol (2006) 63:274–282 DOI: 10.1007/s00239-005-0264-3