Evolutionary history of tissue kallikreins

Abstract

The gene family of human kallikrein-related peptidases (KLKs) encodes proteins with diverse and pleiotropic functions in normal physiology as well as in disease states. Currently, the most widely known KLK is KLK3 or prostate-specific antigen (PSA) that has applications in clinical diagnosis and monitoring of prostate cancer. The KLK gene family encompasses the largest contiguous cluster of serine proteases in humans which is not interrupted by non-KLK genes. This exceptional and unique characteristic of KLKs makes them ideal for evolutionary studies aiming to infer the direction and timing of gene duplication events. Previous studies on the evolution of KLKs were restricted to mammals and the emergence of KLKs was suggested about 150 million years ago (mya). In order to elucidate the evolutionary history of KLKs, we performed comprehensive phylogenetic analyses of KLK homologous proteins in multiple genomes including those that have been completed recently. Interestingly, we were able to identify novel reptilian, avian and amphibian KLK members which allowed us to trace the emergence of KLKs 330 mya. We suggest that a series of duplication and mutation events gave rise to the KLK gene family. The prominent feature of the KLK family is that it consists of tandemly and uninterruptedly arrayed genes in all species under investigation. The chromosomal co-localization in a single cluster distinguishes KLKs from trypsin and other trypsin-like proteases which are spread in different genetic loci. All the defining features of the KLKs were further found to be conserved in the novel KLK protein sequences. The study of this unique family will further assist in selecting new model organisms for functional studies of proteolytic pathways involving KLKs.

Figure 1. Sequence logo of the three catalytic motifs present in KLKs.

The number of each of the motifs is indicated below. The height of each letter is proportional to the frequency of the corresponding residue at that position, and the letters are ordered so the most frequent is on top. The conserved catalytic triad residues are indicated with asterisks. The dot indicates the conserved glycine residue present in the oxyanion hole.

Figure 2. Maximum likelihood tree based on the core domain of the KLK homologues.

The support values (>50%) are indicated at the nodes. The branch lengths depict evolutionary distance. The trypsin proteins are used as outgroup. The scale bar at the upper right denotes evolutionary distance of 0.1 amino acids per position.

Figure 3. Representative maximum likelihood phylogenetic tree of KLK homologues.

Bootstrap values (>50%) are indicated at the nodes. The branch lengths depict evolutionary distance. The trypsin proteins are used as outgroup. The scale bar at the upper right denotes evolutionary distance of 0.2 amino acids per position.

Figure 4. Schematic representation of the chromosomal arrangement of KLKs.

The orientation and approximate position and size of the kallikrein genes. The KLK encoding genes are shown as filled arrowheads, whereas the KLK pseudogenes are represented by open arrowheads. The KLK2, KLK2-ps and KLK3 genes are indicated by red. Loci are drawn in approximate scale. On the left, a NCBI taxonomy-based cladogram shows the evolutionary relationships of taxa and the taxonomic classes.

Figure 5. A, Secondary structure prediction of putative KLKs.

Sequences corresponding to the conserved trypsin domain were aligned using PROMALS3D. The residues of the catalytic triad are shown on the black background; the glycine residue is shown on the gray background. The consensus secondary structure is shown at the bottom, where the α-helices are represented by cylinders and the β-sheets by arrows. KLKs with known crystal structure were used as reference; their PDB accession codes are: KLK1(1spj), KLK5(2psy), KLK6 (1gvl). B, Tertiary structure of proKLK6. Graphic visualization of the human proKLK6 protein color coded by conservation score, where the region in blue corresponds to the most highly conserved region. KLK6 is represented as a cartoon. The four conserved amino acid residues (His ⁵⁷, Asp¹⁰², Ser¹⁹⁵ and Gly¹⁹³) are indicated.

Figure 6. Evolution of KLKs.

Schematic representation of key events occurring through the evolutionary history of KLKs.

Figure 7. Rate shift analysis of frog KLK.

A, Comparison with orphan lizard KLKs and B, Comparison with KLK1. Residues in blue and red background correspond to sites with slower and faster rate of evolution, respectively.