The macrophage receptor with collagenous structure (MARCO) is a cell surface receptor; it plays a significant part in the natural immune response through binding to bacteria by the cysteine-rich structural domain in scavenger receptor (SRCR). Based on our previous data, the full-length cDNA of 1548 bp for MARCO was identified from Larimichthys crocea (named as LcMARCO, PQ842624); its open reading frame was 1218 bp encoding 405 amino acids with a theoretical molecular weight (MW) of 42 kDa and a predicted pI (isoelectric point) of 6.01. BLASTp analysis as well as the phylogenetic relationship indicated that LcMARCO had high similarity to known MARCO from other fish, especially in the SRCR structural domain. After being efficiently expressed in Escherichia coli DE 3 with the expected size, LcMARCO and its SRCR domain were purified by Ni–NTA Superflow resin and were verified to bind to Vibrio alginolyticus, Vibrio harveyi, and Vibrio parahaemolyticus through western blot (WB), respectively. The eukaryotic recombinant vector including MARCO-EGFP was constructed and transfected into human embryonic kidney 293 T (HEK293T) cells for subcellular localization. The results indicated that LcMARCO was expressed as the transmembrane protein mainly on the cell membrane. To tentatively explore the signaling pathway of LcMARCO, ten components of the SRA signaling pathway were identified from L. crocea, including macrophage receptor (MARCO), major vault protein (MVP), protein tyrosine kinase (PTK), Ras-related C3 botulinum toxin substrate 1 (Rac1), serine/threonine-protein kinase PAK 2-like (PAK), ribonuclease P/MRP subunit p38 (Rpp38), interleukin-1 (IL-1), mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinase 2 (ERK2), and tumor necrosis factor alpha (TNF-α), and their expression after being infected by V. parahaemolyticus (1 × 108 CFU/mL) was detected. The results showed that MARCO was initially upregulated at 24 h, earlier than other genes, while the majority of genes exhibited minimal or negligible expression, but MAPK, ERK, and TNF-α increased at 72 h, which drew the outline of the MARCO signal transduction process in L. crocea that consisted of four kinds of elements and their interactions, and its possible path might be MARCO-MVP-PAK-PTK-P38-IL-1 or MARCO-MVP-PAK-MAPK-ERK-TNF-α, and the exact signal path needs our further verification. The above findings indicated that LcMARCO, as a significant transmembrane scavenger receptor, potentially played a crucial role in the immune defense system of L. crocea against pathogens.

Immune mechanism analysis of scavenger receptor MARCO in Larimichthys crocea

期刊论文
发表日期：

2025年6月21日

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Wei Cheng,
Mingming Wenren,
Jiong Wu,
Xu Zhou,
Changfeng Chi,
Zhenming Lü,
Ping Wang,
Huihui Liu

摘要

The macrophage receptor with collagenous structure (MARCO) is a cell surface receptor; it plays a significant part in the natural immune response through binding to bacteria by the cysteine-rich structural domain in scavenger receptor (SRCR). Based on our previous data, the full-length cDNA of 1548 bp for MARCO was identified from Larimichthys crocea (named as LcMARCO, PQ842624); its open reading frame was 1218 bp encoding 405 amino acids with a theoretical molecular weight (MW) of 42 kDa and a predicted pI (isoelectric point) of 6.01. BLASTp analysis as well as the phylogenetic relationship indicated that LcMARCO had high similarity to known MARCO from other fish, especially in the SRCR structural domain. After being efficiently expressed in Escherichia coli DE 3 with the expected size, LcMARCO and its SRCR domain were purified by Ni–NTA Superflow resin and were verified to bind to Vibrio alginolyticus, Vibrio harveyi, and Vibrio parahaemolyticus through western blot (WB), respectively. The eukaryotic recombinant vector including MARCO-EGFP was constructed and transfected into human embryonic kidney 293 T (HEK293T) cells for subcellular localization. The results indicated that LcMARCO was expressed as the transmembrane protein mainly on the cell membrane. To tentatively explore the signaling pathway of LcMARCO, ten components of the SRA signaling pathway were identified from L. crocea, including macrophage receptor (MARCO), major vault protein (MVP), protein tyrosine kinase (PTK), Ras-related C3 botulinum toxin substrate 1 (Rac1), serine/threonine-protein kinase PAK 2-like (PAK), ribonuclease P/MRP subunit p38 (Rpp38), interleukin-1 (IL-1), mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinase 2 (ERK2), and tumor necrosis factor alpha (TNF-α), and their expression after being infected by V. parahaemolyticus (1 × 10⁸ CFU/mL) was detected. The results showed that MARCO was initially upregulated at 24 h, earlier than other genes, while the majority of genes exhibited minimal or negligible expression, but MAPK, ERK, and TNF-α increased at 72 h, which drew the outline of the MARCO signal transduction process in L. crocea that consisted of four kinds of elements and their interactions, and its possible path might be MARCO-MVP-PAK-PTK-P38-IL-1 or MARCO-MVP-PAK-MAPK-ERK-TNF-α, and the exact signal path needs our further verification. The above findings indicated that LcMARCO, as a significant transmembrane scavenger receptor, potentially played a crucial role in the immune defense system of L. crocea against pathogens.