Recombinant Mouse Reticulocalbin-3 (Rcn3)

What is Recombinant Mouse Reticulocalbin-3 and what are its structural characteristics?

Recombinant Mouse Reticulocalbin-3 (Rcn3) is an EF-hand calcium binding domain protein primarily localized in the endoplasmic reticulum. The protein consists of 328 amino acids (Met1-Leu328) in mouse models and is typically expressed with a polyhistidine tag at the C-terminus for purification purposes . Rcn3 belongs to the CREC (Cab45/reticulocalbin/ERC45/calumenin) family of Ca2+-binding proteins containing multiple EF-hand domains. These domains are crucial for its calcium-binding capacity and subsequent biological functions. In experimental settings, recombinant mouse Rcn3 is commonly produced as a fusion protein with tags such as His-tag to facilitate purification and detection. The protein's molecular structure allows it to participate in calcium-dependent processes within the secretory pathway of various cell types, making it a significant factor in cellular calcium homeostasis.

What expression systems are commonly used for producing recombinant Rcn3?

The production of recombinant Rcn3 employs several expression systems, each with distinct advantages depending on research requirements. Human embryonic kidney (HEK-293) cells are frequently utilized for mouse Rcn3 expression, as evidenced by commercial preparations with documented high purity levels exceeding 95% as determined by SDS-PAGE . This mammalian expression system offers proper post-translational modifications and protein folding, which are critical for maintaining biological activity. Alternative expression systems include Escherichia coli (E. coli), which provides cost-effective production but may lack appropriate post-translational modifications, and yeast-based systems that offer a balance between proper protein folding and economical production . The choice of expression system significantly influences protein characteristics such as solubility, activity, and glycosylation patterns. Researchers should select the system based on experimental requirements, considering whether native conformation or specific modifications are essential for their studies.

What are standard methods for detecting Rcn3 in experimental samples?

Detection of Rcn3 in experimental samples employs multiple complementary techniques depending on the research question and sample type. For protein expression analysis, Western blotting serves as a primary method, having been successfully implemented to quantify Rcn3 levels in lung tissues from both COPD patients and mouse models of emphysema . Immunohistochemistry provides spatial information about Rcn3 expression patterns in tissue samples, allowing researchers to identify specific cell types expressing the protein and determine subcellular localization . For transcriptional analysis, quantitative PCR (qPCR) effectively measures Rcn3 mRNA expression, as demonstrated in studies comparing expression levels between control and diseased states . Protein expression can also be analyzed through specialized platforms such as the UALCAN portal, which has been used to profile Rcn3 expression across multiple cancer types . When working with recombinant Rcn3, enzyme-linked immunosorbent assay (ELISA) provides quantitative measurement of protein concentration. Researchers should incorporate appropriate positive and negative controls to validate detection specificity, particularly important when examining tissues with variable expression levels.

How should recombinant Rcn3 be stored and handled in a laboratory setting?

Proper storage and handling of recombinant Rcn3 is crucial for maintaining protein integrity and experimental reproducibility. Recombinant Rcn3 is typically supplied in lyophilized form and should be stored according to manufacturer's recommendations, which often include storage at 4°C for short-term use (2-4 weeks) or at -20°C for long-term preservation . Upon reconstitution, Rcn3 solutions generally contain buffer components that stabilize the protein, such as Tris-HCl (pH 8.0), DTT, glycerol, and NaCl . For optimal stability during long-term storage, addition of carrier proteins (0.1% HSA or BSA) is recommended to prevent protein adsorption to storage vessels and maintain activity . Multiple freeze-thaw cycles should be strictly avoided as they can significantly compromise protein integrity through denaturation and aggregation. Working aliquots should be prepared upon initial reconstitution to minimize repeated freezing and thawing. When handling the protein for experiments, researchers should consider maintaining consistent buffer conditions to prevent pH or ionic strength fluctuations that could affect protein function. Documentation of storage conditions, reconstitution dates, and freeze-thaw history is essential for experimental reproducibility.

What is the role of Rcn3 in lung development and pathogenesis of COPD?

Rcn3 plays a significant role in both lung development and the pathogenesis of COPD, with evidence suggesting differential regulatory functions depending on the physiological context. During perinatal lung development, Rcn3 in type II alveolar epithelial cells (AECIIs) functions as a critical regulator of alveolarization and surfactant metabolism . Intriguingly, in the context of COPD, Rcn3 expression is significantly upregulated in lung specimens from patients compared to non-COPD controls, with expression levels positively correlating with the severity of emphysema as measured by mean linear intercept (MLI) . This pattern was consistently observed in mouse models of emphysema induced by either cigarette smoke exposure or elastase administration, where both Rcn3 protein and mRNA levels were markedly increased . The selective ablation of Rcn3 in AECIIs using conditional knockout (CKO) mice significantly alleviated the severity of elastase-induced emphysema, suggesting that Rcn3 overexpression may contribute to disease progression rather than represent a compensatory response . Mechanistically, Rcn3 may influence COPD pathogenesis through multiple pathways, including regulation of ER stress responses, modulation of calcium-dependent signaling in alveolar cells, and potential effects on the protease-antiprotease balance that underpins the currently accepted hypothesis of emphysema development. These findings collectively position Rcn3 as both a potential biomarker for COPD progression and a promising therapeutic target for intervention strategies.

How does Rcn3 influence collagen production and cardiac fibrosis?

Rcn3 exerts significant regulatory control over collagen production, functioning as a novel negative regulator in the context of cardiac fibrosis. Differential proteomics analysis identified the reticulocalbin family members, including Rcn3, as proteins commonly modified by profibrotic stimuli such as Aldosterone (Aldo), Galectin-3 (Gal-3), and Cardiotrophin-1 (CT-1) in human cardiac fibroblasts . Notably, these profibrotic agents all triggered downregulation of Rcn3 expression, suggesting an inverse relationship between Rcn3 levels and fibrotic responses . Direct experimental evidence demonstrated that treatment with recombinant Rcn3 decreased collagen expression in human cardiac fibroblasts through modulation of the Akt phosphorylation pathway . Furthermore, CRISPR/Cas9-mediated activation of endogenous Rcn3 expression resulted in reduced collagen production, confirming the antifibrotic role of Rcn3 through both exogenous application and genetic upregulation approaches . The protective effects of recombinant Rcn3 extended beyond a single profibrotic stimulus, as it effectively blocked collagen expression induced by multiple agents including Aldosterone, Galectin-3, Cardiotrophin-1, and Angiotensin II . These findings collectively establish Rcn3 as a potential therapeutic target for cardiac fibrosis intervention, where strategies aimed at increasing Rcn3 expression or activity could counteract pathological collagen accumulation. The mechanistic insights into how Rcn3 regulates Akt phosphorylation provide a molecular framework for understanding its antifibrotic actions and developing targeted interventions for fibrotic cardiac diseases.

What is the relationship between Rcn3 expression and cancer development?

The relationship between Rcn3 expression and cancer development demonstrates complex patterns across different malignancies, suggesting context-dependent oncogenic and tumor-suppressive roles. Pan-cancer analysis has revealed significant variation in Rcn3 genetic alterations across cancer types, with the highest alteration frequency (>4%) observed in uterine cancers, where mutations represent the predominant alteration type . In contrast, adrenocortical carcinoma (ACC), breast invasive carcinoma, and pancreatic tumors predominantly exhibit copy number amplification of Rcn3, suggesting potential oncogenic functions in these contexts . Significant positive correlations between Rcn3 expression and copy number alterations (CNA) have been documented in multiple cancer types, including rectal adenocarcinoma (READ), sarcoma (SARC), uterine corpus endometrial carcinoma (UCEC), and others, indicating that genomic amplification contributes to increased Rcn3 expression . Epigenetic regulation through DNA methylation further influences Rcn3 expression, with significant negative correlations between expression and promoter methylation observed across numerous cancer types including bladder, breast, cervical, and lung cancers . The differential expression patterns of Rcn3 across cancer stages suggest potential roles in disease progression, while survival analyses provide insights into its prognostic significance. Protein expression analysis through platforms like UALCAN has further validated differential Rcn3 expression between normal and tumor tissues in breast, colon, ovarian, renal, and endometrial cancers . These multifaceted relationships between Rcn3 and cancer highlight its potential utility as both a biomarker and therapeutic target, though further mechanistic studies are needed to clarify its precise functions in different cancer contexts.