Leucine-rich KQLLL Motif Coordinates Secretion, Nuclear Localization, and Proliferation of Chitinase 3-like 1

Chitinase 3-like-1 (CHI3L1/YKL-40) is a secreted glycoprotein that binds to chitin but lacks enzymatic activity. Recent studies have suggested that CHI3L1 nuclear localization is associated with the transition from inflammation to carcinogenesis. To investigate the mechanism underlying CHI3L1 nuclear translocation, we analyzed its putative nuclear localization signals (NLSs) using cNLS Mapper and identified 9 putative NLS sequences. The region with the highest scoring (amino acids 144-173) contains a leucine-rich KQLLL motif at its C-terminal end (residues 170-174). This study aimed to clarify the functional role of this motif.

SW480 human colorectal cancer cells (CRCs) with KQLLL motif deletion (KO cells) were generated using the CRISPR-Cas9 system. CHI3L1 expression and secretion were evaluated by western blotting and enzyme-linked immunosorbent assay, respectively. The subcellular localization of CHI3L1 with or without TNF-α stimulation, was examined by immunofluorescence analysis. Cell proliferation was assessed using the MTS assay.

CHI3L1 secretion was almost undetectable in KO cells, accompanied by a marked reduction in intracellular expression. Immunofluorescence analysis demonstrated partial nuclear localization of CHI3L1 in wild-type (WT) cells, with predominant cytoplasmic and perinuclear distribution. In contrast, KO cells showed markedly diminished CHI3L1 signals. Functionally, KO cells exhibited significantly reduced proliferation compared with WT cells (p<0.01).

The KQLLL motif regulates CHI3L1 expression, secretion, and subcellular localization, suggesting a critical role in CRC cells proliferation.