Alternative splicing is critical for the expansion of proteomic diversity and the regulation of gene expression. It is also one of the major mechanisms in the genome for the creation of new proteins during evolution. The human brain, widely accepted as a product of adaptive evolution, preferably utilizes this strategy in its functional complexity. Hence, identifying human-specific alternative splicing forms in central nervous system (CNS) is thus important for understanding the mechanisms of functional evolution of human cognition. KLK8 (Kallikrein 8, or called neuropsin) is a serine protease functioning in CNS involved in learning and memory. Previous studies show that its splicing pattern is different between human and mouse, with the longer form (type Ⅱ) only expressed in human. Sequence analysis also suggested a recent origin of type Ⅱ during primate evolution. In this study, we aim to understand 1) the mechanism of the creation of the novel form KLK8 type Ⅱduring human brain evolution, 2) the new functional character of the novel form compared to original protein isoform (KLK8 type Ⅰ). We find that the type Ⅱis absent in nonhuman primates, and is thus a human-specific splice form. With the use of an in vitro splicing assay, we show that a human-specific T to A mutation triggers the changes of splicing pattern, leading to the origin of a novel splice form in human brain. Using mutation assay, we prove that this mutation is not only necessary but also sufficient for type Ⅱ expression. In addition, using mutagenesis we also prove that multiple sites weaken KLK8’s original constitutive splicing. It is likely that both the creation of novel splice form and the weakening of constitutive splicing contribute to the splicing pattern changes during primate evolution, which suggests a mutistep process eventually leading to the origin of the typeⅡform in human. 5’RACE assay, promoter sequence analysis and promoter activity experiment indicate that transcription regulation of KLK8 is a dynamic process during evolution. Our results demonstrate a molecular mechanism for the creation of novel proteins through alternative splicing in CNS during human evolution. Additionally, RT-PCR and Western blot show that this novel form is expressed in a temporal-spatial manner and secretion efficiency is cell-type dependent. Biochemical assays and enzyme activity experiment indicate that KLK8 typeⅡnot only can produce the active form of KLK8, but also contains a type Ⅱ-specific intermediate protein form, which suggests that the emergence of type Ⅱ KLK8 in the human brain likely leads to functional modifications of KLK8.
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