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Type II) Form of Kallikrein 8, a Gene Involved in Learning and Memory

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Type II) Form of Kallikrein 8, a Gene Involved in Learning and Memory Cell Research (2009) 19:259-267. npg © 2009 IBCB, SIBS, CAS All rights reserved 1001-0602/09 $ 30.00 ORIGINAL ARTICLE www.nature.com/cr Functional characterization of the human-specific (type II) form of kallikrein 8, a gene involved in learning and memory Zhi-xiang Lu1, 2, 4, Qin Huang3, 4, Bing Su1, 2 1State Key Laboratory of Genetic Resources and Evolution, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming 650223, China; 2Kunming Primate Research Center, Chinese Academy of Sciences, Kunming 650223, China; 3Institute of Bio- chemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China; 4Graduate School of Chinese Academy of Sciences, Beijing 100049, China Kallikrein 8 (KLK8) is a serine protease functioning in the central nervous system, and essential in many aspects of neuronal activities. Sequence comparison and gene expression analysis among diverse primate species identified a human-specific splice form of KLK8 (type II) with preferential expression in the human brain, which may contribute to the origin of human cognition. To gain insights into the physiological and biochemical role of this novel form, we conducted functional analyses of human type II KLK8. Our results show that type II KLK8 is abundantly expressed in human embryonic stem cells and in embryo brain samples, suggesting a potential role in embryogenesis. There are dramatic expression variations in different individuals and brain regions, which is a reflection of its dynamic role in neural activities. Furthermore, the transcription start site (TSS) of KLK8 is tissue-specific, with a brain-specific TSS found in humans indicating functional specialization. Our in vitro biochemical assay shows that there is a type II- specific intermediate protein form, although the processed end-point enzymes are the same for both type I and type II KLK8, suggesting that the emergence of type II KLK8 in the human brain likely leads to functional modifications of KLK8. Keywords: type II KLK8, alternative splicing, cognition, human evolution Cell Research (2009) 19:259-267. doi: 10.1038/cr.2009.4; published online 6 January 2009 Introduction abilities are among the most significant changes during the origin of our own species. Hence, elucidating the Alternative splicing (AS) is critical for the expansion functional consequences of human-specific splice forms of proteomic diversity and the regulation of gene expres- in the central nervous system (CNS) is a prerequisite for sion [1]. It is also one of the major mechanisms in the improved understanding of the functional evolution of genome for the creation of new proteins during evolution human cognition. [2, 3]. The human brain, widely accepted as a product KLK8 (also called neuropsin, MIM# 605644) is a of adaptive evolution, preferably utilizes this strategy in secreted-type serine protease preferentially expressed in its functional complexity [4]. Experimental studies of CNS, and was initially cloned from the mouse hippocam- individual genes have revealed many cases of functional pus [7]. Electrophysiology and behavioral studies have differences between different splice forms during onto- implicated its involvement in hippocampal plasticity and genesis. Sometimes, the ratio change of the expressed kindling epileptogenesis [8-10]. splice variants may have pathogenic consequences [5, Human KLK8 was originally cloned as the human 6]. The enlarged brain and highly improved cognitive ortholog of the mouse brain protease [11] and is known to be associated with diseases such as ovarian cancer and Alzheimer’s disease [12-14]. It is also a favorable Correspondence: Bing Su prognostic marker in clinical diagnosis [15, 16]. This Tel: +86-871-5120212 gene is alternatively spliced in the human brain. Mitsui E-mail: [email protected] Received 27 June 2008; revised 30 June 2008; accepted 30 June 2008; et al. [17] reported two types of human KLK8 cDNAs in published online 6 January 2009 the brain. One of them (type I) is homologous with the npg Functional characterization of human-specific KLK8 260 mouse counterpart while the other (type II) is absent in The in vitro biochemical assay reveals that type I and the mouse (Figure 1). The two transcripts differ only in type II forms are released in a cell type-dependent man- their exon 3 sequences. Type II includes extra 45 amino ner. Additionally, type II is more efficiently secreted in acids at the N-terminus of exon 3 due to alternative splic- High Five cells, though the final active proteins are the ing. In vitro splicing assay has shown that a human-spe- same for both type I and type II KLK8, suggesting func- cific point mutation led to the origin of this novel form tional modifications for type II in the human brain. in the human brain. Moreover, the chimpanzee sequence was able to express type II KLK8 when the human-like Results mutation was introduced [18]. Similar to type I, human- specific type II KLK8 is also abundantly expressed in the Temporal-spatial expression of type II KLK8 in human CNS of both fetuses and adults, as well as in the placenta brain [17, 18]. However, little is known about the biological Our earlier studies using in vitro splicing assays have function of type II KLK8, even though earlier studies demonstrated that generation of type II KLK8, which have proposed biochemical and enzymatic properties for is caused by a single nucleotide substitution, was fixed human type I KLK8 [19, 20]. during human evolution and is therefore absent in non- With the use of RT-PCR and parallel biochemical human primates [18]. To further understand the detailed assays, we show that the expression of human type II expression pattern of type II we conducted a study, in KLK8 starts during embryogenesis and that there are which different developmental stages and different brain large inter-individual and inter-brain region variations. regions in humans were examined. A total of three em- Site1 h_KLK8_type I 23 h_KLK8_type II 60 m_KLK8 23 Site2 h_KLK8_type I 75 h_KLK8_type II 120 m_KLK8 75 h_KLK8_type I 135 h_KLK8_type II 180 m_KLK8 135 h_KLK8_type I 195 h_KLK8_type II 240 m_KLK8 195 h_KLK8_type I 255 h_KLK8_type II 300 m_KLK8 255 h_KLK8_type I IGSKG 260 Type II specific extra 45 amino acids h_KLK8_type II IGSKG 305 m_KLK8 MDNRD 260 Conserved triplet of serine protease Figure 1 Alignment of protein sequences of human and mouse KLK8. Site 1 and Site 2 refer to the endoprotease sites. KLK8 activation occurs after endoprotease cleaves next to the lysine residue at the site 2. Bold letters indicate species-specific secretion signal peptides of KLK8. The human sequence exhibits a 23 amino acid signal peptide, while the mouse sequence possesses a 28 amino acid signal peptide [19, 32]. Cell Research | Vol 19 No 2 | February 2009 Zhi-xiang Lu et al. npg 261 bryos and nine adult brain tissue samples were tested and Brain-specific TSS in KLK8 expression a complex expression pattern was observed for type II Recent reports have shown that gene expression can (Figure 2A and 2B). For example, of the three embryo be regulated by alternative UTRs [26, 27], which could brain samples tested, two expressed type II (HE02 and also affect alternative splicing through differential pro- HE03) and the other lacked its expression (HE01). In the moter usage [28-30]. Our earlier data have suggested the nine adult brain samples examined, only four showed divergence of KLK8 promoter sequences and that KLK8 type II expression. In addition to the inter-individual TSS differs between human and non-human primate variation, type II is also characterized by inter-regional brains [18]. With the use of 5′ RACE, we determined variations in brain expression. For example, in embryo the TSS for both type I and type II KLK8 in different tis- brain sample, HE03 expression was detected in the mid- sue samples from human and rhesus macaque (Macaca brain and posterior brain, but absent from the frontal mulatta) (Figure 3). Although type I and type II share the brain. Similar to its ancestral type (type I) [8, 21-23], same TSS in humans and lack a splice-form-specific pro- type II was also expressed in a temporal-spatial manner. moter, the brain TSSs in humans and rhesus macaques We also tested human embryonic stem cells and observed differ [18] and exhibit tissue specificity. This observa- an abundance of type II expression, suggesting a poten- tion suggests that during the origin of type II, there was tial role in early embryogenesis (Figure 2C). a brain-specific expression regulation change, although type I KLK8 is commonly expressed in many tissues un- Type II KLK8 transcript can produce an intact protein in der normal and pathological conditions [31]. vitro Structural modeling of splice variants from genes of Similar secretion and activation efficiency between type I 1% of the genome surveyed by the ENCODE consortium and type II KLK8 has predicted that many alternative splicing events are Earlier studies have demonstrated that in vivo acti- unlikely to result in correct protein folding [24], which vation of KLK8 includes two key steps. First, the N- may lead to instability, degradation and malfunction [25]. terminus signal peptide (23 amino acids in humans [20]; To prove that the novel form of human KLK8 could be 28 amino acids in mice [32]) (Figure 1) is removed after normally translated, we cloned the full length coding KLK8 is translocated into the endoplasmic reticulum sequence of KLK8. Western blot analysis detected two in the host cell (from pre-pro-protein to pro-protein). bands corresponding to type I and type II KLK8, respec- Second, the pro-protein is enzymatically processed by tively (Figure 2D), indicating that type II KLK8 is likely endoprotease (site 1 and 2 in Figure 1) and becomes to be produced as an intact protein.
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