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A Heparin Binding Motif Rich in Arginine and Lysine is the Functional Domain of YKL-40.

ABSTRACT: The heparin-binding glycoprotein YKL-40 (CHI3L1) is intimately associated with microvascularization in multiple human diseases including cancer and inflammation. However, the heparin-binding domain(s) pertinent to the angiogenic activity have yet been identified. YKL-40 harbors a consensus heparin-binding motif that consists of positively charged arginine (R) and lysine (K) (RRDK; residues 144-147); but they don't bind to heparin. Intriguingly, we identified a separate KR-rich domain (residues 334-345) that does display strong heparin binding affinity. A short synthetic peptide spanning this KR-rich domain successfully competed with YKL-40 and blocked its ability to bind heparin. Three individual point mutations, where alanine (A) substituted for K or R (K337A, K342A, R344A), led to remarkable decreases in heparin-binding ability and angiogenic activity. In addition, a neutralizing anti-YKL-40 antibody that targets these residues and prevents heparin binding impeded angiogenesis in vitro. MDA-MB-231 breast cancer cells engineered to express ectopic K337A, K342A or R344A mutants displayed reduced tumor development and compromised tumor vessel formation in mice relative to control cells expressing wild-type YKL-40. These data reveal that the KR-rich heparin-binding motif is the functional heparin-binding domain of YKL-40. Our findings shed light on novel molecular mechanisms underlying endothelial cell angiogenesis promoted by YKL-40 in a variety of diseases.

SUBMITTER: Ngernyuang N

PROVIDER: S-EPMC5773473 | biostudies-literature | 2018 Feb

REPOSITORIES: biostudies-literature

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A Heparin Binding Motif Rich in Arginine and Lysine is the Functional Domain of YKL-40.

Ngernyuang Nipaporn N Yan Wei W Schwartz Lawrence M LM Oh Dennis D Liu Ying-Bin YB Chen Hongzhuan H Shao Rong R

Neoplasia (New York, N.Y.) 20171220 2

The heparin-binding glycoprotein YKL-40 (CHI3L1) is intimately associated with microvascularization in multiple human diseases including cancer and inflammation. However, the heparin-binding domain(s) pertinent to the angiogenic activity have yet been identified. YKL-40 harbors a consensus heparin-binding motif that consists of positively charged arginine (R) and lysine (K) (RRDK; residues 144-147); but they don't bind to heparin. Intriguingly, we identified a separate KR-rich domain (residues 3 ...[more]

PMID: 29274508

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Project description:Bacteriophage ? N protein binds boxB RNA hairpins in the nut (N utilization) sites of immediate early ? transcripts and interacts with host factors to suppress transcriptional termination at downstream terminators. In opposition to ? N, the Nun protein of HK022 binds the boxBs of coinfecting ? transcripts, interacts with a similar or identical set of host factors, and terminates transcription to suppress ? replication. Comparison of N-boxB and Nun-boxB nuclear magnetic resonance (NMR) structural models suggests similar interactions, though limited mutagenesis of Nun is available. Here, libraries of Nun's arginine-rich motif (ARM) were screened for the ability to exclude ? coinfection, and mutants were assayed for Nun termination with a boxB plasmid reporter system. Several Nun ARM residues appear to be immutable: Asp26, Arg28, Arg29, Arg32, Trp33, and Arg36. Asp26 and Trp33 appear to be unable to contact boxB and are not found at equivalent positions in ? N ARM. To understand if the requirement of Asp26, Trp33, and Arg36 indicated differences between HK022 Nun termination and ? N antitermination complexes, the same Nun libraries were fused to the activation domain of ? N and screened for clones able to complement N-deficient ?. Mutants were assayed for N antitermination. Surprisingly, Asp26 and Trp33 were still essential when Nun ARM was fused to N. Docking suggests that Nun ARM contacts a hydrophobic surface of the NusG carboxy-terminal domain containing residues necessary for Nun function. These findings indicate that Nun ARM relies on distinct contacts in its ternary complex and illustrate how protein-RNA recognition can evolve new regulatory functions.? N protein interacts with host factors to allow ? nut-containing transcripts to elongate past termination signals. A competing bacteriophage, HK022, expresses Nun protein, which causes termination of ? nut transcripts. ? N and HK022 Nun use similar arginine-rich motifs (ARMs) to bind the same boxB RNAs in nut transcripts. Screening libraries of Nun ARM mutants, both in HK022 Nun and in a ? N fusion, revealed amino acids essential to Nun that could bind one or more host factors. Docking suggests that NusG, which is present in both Nun termination and N antitermination, is a plausible partner. These findings could help understand how transcription elongation is regulated and illustrate how subtle differences allow ARMs to evolve new regulatory functions.

Dataset Information

A Heparin Binding Motif Rich in Arginine and Lysine is the Functional Domain of YKL-40.

Publications

A Heparin Binding Motif Rich in Arginine and Lysine is the Functional Domain of YKL-40.

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