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Proximity labeling and identification of the proteins that interact with Siglec-14 on THP-1 cells

ABSTRACT: Proteins that interact with Siglec-14 were identified by proximity labeling, followed by streptavidin affinity purification and proteomic analysis. MaxQuant software was used for the quantitative analysis of the proteins identified.

INSTRUMENT(S): LTQ Orbitrap Velos

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Monocyte, Cell Culture

SUBMITTER: Jimmy Huang

LAB HEAD: Takashi Angata

PROVIDER: PXD009748 | Pride | 2018-11-04

REPOSITORIES: Pride

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			Action	DRS
	EV_0928_171011-2.raw	Raw
	EV_1003_171011.raw	Raw
	F-sig14_0928_171011.raw	Raw
	F-sig14_1003_171011.raw	Raw
	MaxQuant_Output.zip	Other

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Publications

Soluble Siglec-14 glycan-recognition protein is generated by alternative splicing and suppresses myeloid inflammatory responses.

Huang Po-Chun Jimmy PJ Low Penk-Yeir PY Wang Iren I Hsu Shang-Te Danny SD Angata Takashi T

The Journal of biological chemistry 20181030 51

Human sialic acid-binding immunoglobulin-like lectin 14 (Siglec-14) is a glycan-recognition protein that is expressed on myeloid cells, recognizes bacterial pathogens, and elicits pro-inflammatory responses. Although Siglec-14 is a transmembrane protein, a soluble form of Siglec-14 is also present in human blood. However, the mechanism that generates soluble Siglec-14 and what role this protein form may play remain unknown. Here, investigating the generation and function of soluble Siglec-14, we ...[more]

PMID: 30377253

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Identification of Siglec-7-ligand candidates on JVM-3 human B cell line by proximity labeling

Project description:Siglec-7 is a member of Siglec family of glycan recognition proteins involved in the regulation of natural killer (NK) cell activities. We found Siglec-7 ligand is highly expressed on JVM-3, a B cell chronic lymphocytic leukemia (B-CLL) cell line. In this experiment, we attempted at identifying the ligands of Siglec-7 on JVM-3 by applying the proximity labeling method we developed (Chang L et al., J Proteome Res. 2017, 16:3929-41).

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Identification of Siglec-E-ligand candidates on BCL-1 mouse B cell line by proximity labeling

Project description:Siglec-E is a member of Siglec family of glycan recognition proteins involved in the regulation of myeloid cell activities. Siglec-E preferentially recognizes disialic acid, a glycan epitope consisting of two sialic acids conjugated in alpha2-8 linkage (Zhang JQ et al., Eur J Immunol. 2004, 34:1175-84). BCL-1, a mouse B cell line, expresses disialic acid epitope (Wang SH et al., Glycobiology. 2013, 23:677-89). In this experiment, we attempted at identifying the ligands of Siglec-E on BCL-1 by applying the proximity labeling method we developed (Chang L et al., J Proteome Res. 2017, 16:3929-41).

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Identification of Siglec ligands in human B cell

Project description:Siglecs are a family of receptor-type glycan recognition proteins (lectins) involved in self - nonself discrimination by the immune system. Identification of Siglec ligand(s) is necessary to understand how Siglec-ligand interaction translates to biological outcomes, but is challenging because the interaction is weak. To facilitate identification of Siglec ligands, we adopted proximity labeling method based on tyramide radicalization principle.

2017-09-29 | PXD006359 | Pride

Structural insights into Siglec-15 reveal glycosylation dependency for its interaction with T cells through integrin CD11b

Project description:Sialic acid-binding Ig-like lectin 15 (Siglec-15) is emerging as an immune modulator and target for cancer immunotherapy. However, limited knowledge regarding its mechanism of action and structure, or its binding partners with T cells, restrains the development of drug candidates that could unleash its full therapeutic potential. Here, we determine the crystal structure of Siglec-15 and delineate its binding epitope by co-crystallization with an anti-Siglec-15 blocking antibody. Saturation transfer-difference nuclear magnetic resonance (STD-NMR) spectroscopy assisted with molecular dynamics simulations reveals the binding mode of Siglec-15 to (2,3)- and 2,6)- linked sialic acids and to the cancer-associated sialyl-Tn (STn) glycoform. Binding of Siglec-15 to T cells, which lack STn expression, depends on the presence of (2,3)- and 2,6)- linked sialoglycans. We identify the leukocyte integrin CD11b as a binding partner of Siglec-15 on human T cells. Collectively, these data provide an integrative understanding of the structural features of Siglec-15 and underscore glycosylation as an additional layer of control of T cell responses.

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Proximity labeling of Siglec-1 counter-receptors on activated regulatory T cells using soluble Siglec-1-HRP multimers

Project description:Siglec-1 is a macrophage lectin-like receptor that mediates sialic acid-dependent cellular interactions. It was shown previously to promote inflammation in autoimmune disease through suppressing the expansion of regulatory T cells (Tregs). We have investigated the molecular basis for Siglec-1 binding to these cells using in vitro-induced Tregs. A proximity labelling and proteomics strategy were used to identify glycoproteins expressed by activated Tregs that may function as Siglec-1 counter-receptors.

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Gene expression profiles of Siglec-14/THP-1 and Siglec-5/THP cell lines, with or without NTHi stimulation

Project description:Acquisition of a new strain of non-typeable Haemophilus influenzae (NTHi) is often associated with exacerbation of chronic obstructive pulmonary disease (COPD). We have previously reported that COPD patients who are homozygous null for SIGLEC14 gene is less susceptible to COPD exacerbation than those who have wild-type allele with functional SIGLEC14 gene. In order to gain insight into the mechanism behind the COPD exacerbation, and to find new clues that may lead to the discovery of objective biomarker of COPD exacerbation, Siglec-14/THP-1 and Siglec-5/THP-1 cell lines, which mimic monocytes from homozygous wild-type and homozygous SIGLEC14-null person, respectively, were incubated with or without NTHi, and their gene expression profiles were compared by using Affymetrix Human Genome U133 Plus 2.0 Array. Four samples (2 cell lines x 2 conditions) were analyzed. No replicates were made.

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Identification of proteins that interact with Siglec-6

Project description:Siglec-6 is a glycoprotein overexpressed on chronic lymphocytic leukemia (CLL) cell. We have shown that Siglec-6 promotes migration and adhesion of CLL cells towards CLL bone marrow stromal cells. To elucidate the mechanistic pathway involved in Siglec-6 dependent migration, we performed mass spectrometry proteomics analysis on MEC1-002 CLL cell line pulled down with a Siglec-6 targeted antibody, which revealed that Siglec-6 interacts with DOCK8, a guanine nucleotide exchange factor. Interaction of Siglec-6 with its ligand promotes DOCK8 dependent Cdc42 activation, WASP protein recruitment and F-actin polymerization. Therapeutically, Siglec-6 leukemic cells can be eliminated with a Siglec-6 targeted bispecific antibody in vitro and in vivo.

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Patient-derived anti-Siglec-6 antibodies engineered into T cell-recruiting bispecific antibody formats for treating leukemia

Project description:Despite numerous therapeutic options, safe and curative therapy is out of reach for most chronic lymphocytic leukemia (CLL) patients. We previously reported the identification of a patient-derived CLL-binding antibody, JML-1, and identified Siglec-6 as the target of JML-1. Siglec-6 is an attractive target for cancer immunotherapy due to its absence on healthy tissues and potential role in immune signaling. In this work, we identify additional patient-derived anti-Siglec-6 antibodies, RC-1 and RC-2, that bind with higher affinity than JML-1, yet exhibit similar specificity and overlapping conformational epitopes. Both JML-1 and RC-1 were employed to generate anti-Siglec-6 x anti-CD3 T cell-recruiting bispecific antibodies (T-biAbs) which were effective in vitro at activating T cells and lysing target cells. Upon engineering RC-1 into more compact and rigid T-biAb formats, we achieved sub-pM EC50 values for cell lysis in vitro. In addition, the T-biAbs mediated the killing of primary CLL cells by autologous T cells at natural effector-to-target cell ratios ex vivo. The increased potency of this T-biAb format appears to be due to the reduction in the length and/or flexibility of the cytolytic synapse. Furthermore, the anti-Siglec-6 x anti-CD3 T-biAb was effective at curing mice in a systemic in vivo model of CLL.

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