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Structural and functional characterization of β₂ -glycoprotein I domain 1 in anti-melanoma cell migration.

ABSTRACT: We previously found that circulating β₂ -glycoprotein I inhibits human endothelial cell migration, proliferation, and angiogenesis by diverse mechanisms. In the present study, we investigated the antitumor activities of β₂ -glycoprotein I using structure-function analysis and mapped the critical region within the β₂ -glycoprotein I peptide sequence that mediates anticancer effects. We constructed recombinant cDNA and purified different β₂ -glycoprotein I polypeptide domains using a baculovirus expression system. We found that purified β₂ -glycoprotein I, as well as recombinant β₂ -glycoprotein I full-length (D12345), polypeptide domains I-IV (D1234), and polypeptide domain I (D1) significantly inhibited melanoma cell migration, proliferation and invasion. Western blot analyses were used to determine the dysregulated expression of proteins essential for intracellular signaling pathways in B16-F10 treated with β₂ -glycoprotein I and variant recombinant polypeptides. Using a melanoma mouse model, we found that D1 polypeptide showed stronger potency in suppressing tumor growth. Structural analysis showed that fragments A and B within domain I would be the critical regions responsible for antitumor activity. Annexin A2 was identified as the counterpart molecule for β₂ -glycoprotein I by immunofluorescence and coimmunoprecipitation assays. Interaction between specific amino acids of β₂ -glycoprotein I D1 and annexin A2 was later evaluated by the molecular docking approach. Moreover, five amino acid residues were selected from fragments A and B for functional evaluation using site-directed mutagenesis, and P11A, M42A, and I55P mutations were shown to disrupt the anti-melanoma cell migration ability of β₂ -glycoprotein I. This is the first study to show the therapeutic potential of β₂ -glycoprotein I D1 in the treatment of melanoma progression.

SUBMITTER: Leu SJ

PROVIDER: S-EPMC6549912 | biostudies-literature | 2019 Jun

REPOSITORIES: biostudies-literature

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Structural and functional characterization of β2 -glycoprotein I domain 1 in anti-melanoma cell migration.

Leu Shr-Jeng Jim SJ Lee Tzong-Yi TY Cheng Shu-Wei SW Tsai Meng-Ying MY Lin Yu-Shan YS Chiou Tzeon-Jye TJ Huang Kai-Yao KY Chiang An-Na AN

Cancer science 20190515 6

We previously found that circulating β2 -glycoprotein I inhibits human endothelial cell migration, proliferation, and angiogenesis by diverse mechanisms. In the present study, we investigated the antitumor activities of β2 -glycoprotein I using structure-function analysis and mapped the critical region within the β2 -glycoprotein I peptide sequence that mediates anticancer effects. We constructed recombinant cDNA and purified different β2 -glycoprotein ...[more]

PMID: 31012976

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Project description:The entry of the SARS coronavirus (SCV) into cells is initiated by binding of its spike envelope glycoprotein (S) to a receptor, ACE2. We and others identified the receptor-binding domain (RBD) by using S fragments of various lengths but all including the amino acid residue 318 and two other potential glycosylation sites. To further characterize the role of glycosylation and identify residues important for its function as an interacting partner of ACE2, we have cloned, expressed and characterized various soluble fragments of S containing RBD, and mutated all potential glycosylation sites and 32 other residues. The shortest of these fragments still able to bind the receptor ACE2 did not include residue 318 (which is a potential glycosylation site), but started at residue 319, and has only two potential glycosylation sites (residues 330 and 357). Mutation of each of these sites to either alanine or glutamine, as well as mutation of residue 318 to alanine in longer fragments resulted in the same decrease of molecular weight (by approximately 3 kDa) suggesting that all glycosylation sites are functional. Simultaneous mutation of all glycosylation sites resulted in lack of expression suggesting that at least one glycosylation site (any of the three) is required for expression. Glycosylation did not affect binding to ACE2. Alanine scanning mutagenesis of the fragment S319-518 resulted in the identification of ten residues (K390, R426, D429, T431, I455, N473, F483, Q492, Y494, R495) that significantly reduced binding to ACE2, and one residue (D393) that appears to increase binding. Mutation of residue T431 reduced binding by about 2-fold, and mutation of the other eight residues--by more than 10-fold. Analysis of these data and the mapping of these mutations on the recently determined crystal structure of a fragment containing the RBD complexed to ACE2 (Li, F, Li, W, Farzan, M, and Harrison, S. C., submitted) suggested the existence of two hot spots on the S RBD surface, R426 and N473, which are likely to contribute significant portion of the binding energy. The finding that most of the mutations (23 out of 34 including glycosylation sites) do not affect the RBD binding function indicates possible mechanisms for evasion of immune responses.

Project description:RationaleThe cardiac sodium channel NaV1.5 has a fundamental role in excitability and conduction. Previous studies have shown that sodium channels cluster together in specific cellular subdomains. Their association with intracellular organelles in defined regions of the myocytes, and the functional consequences of that association, remain to be defined.ObjectiveTo characterize a subcellular domain formed by sodium channel clusters in the crest region of the myocytes and the subjacent subsarcolemmal mitochondria.Methods and resultsThrough a combination of imaging approaches including super-resolution microscopy and electron microscopy we identified, in adult cardiac myocytes, a NaV1.5 subpopulation in close proximity to subjacent subsarcolemmal mitochondria; we further found that subjacent subsarcolemmal mitochondria preferentially host the mitochondrial NCLX (Na+/Ca2+ exchanger). This anatomic proximity led us to investigate functional changes in mitochondria resulting from sodium channel activity. Upon TTX (tetrodotoxin) exposure, mitochondria near NaV1.5 channels accumulated more Ca2+ and showed increased reactive oxygen species production when compared with interfibrillar mitochondria. Finally, crosstalk between NaV1.5 channels and mitochondria was analyzed at a transcriptional level. We found that SCN5A (encoding NaV1.5) and SLC8B1 (which encode NaV1.5 and NCLX, respectively) are negatively correlated both in a human transcriptome data set (Genotype-Tissue Expression) and in human-induced pluripotent stem cell-derived cardiac myocytes deficient in SCN5A.ConclusionsWe describe an anatomic hub (a couplon) formed by sodium channel clusters and subjacent subsarcolemmal mitochondria. Preferential localization of NCLX to this domain allows for functional coupling where the extrusion of Ca2+ from the mitochondria is powered, at least in part, by the entry of sodium through NaV1.5 channels. These results provide a novel entry-point into a mechanistic understanding of the intersection between electrical and structural functions of the heart.

Dataset Information

Structural and functional characterization of β₂ -glycoprotein I domain 1 in anti-melanoma cell migration.

Publications

Structural and functional characterization of β<sub>2</sub> -glycoprotein I domain 1 in anti-melanoma cell migration.

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