PGLYRP1 Human

What is PGLYRP1 and how is it structurally characterized?

PGLYRP1, also known as PGRP-S or Tag-7, is a 28 kDa secreted glycoprotein that belongs to the peptidoglycan recognition protein family. The mature human PGLYRP1 is 175 amino acids in length (spanning from Gln22 to Pro196) and contains three variably-sized peptide-carbohydrate recognition sequences of 15, 29, and 49 amino acids, respectively. The protein contains at least three highly conserved C-terminal PGRP domains that are preserved from insects to mammals .

Structurally, recombinant human PGLYRP1 forms homodimers. When analyzed by SDS-gel electrophoresis under reducing conditions (with 5 mM β-mercaptoethanol), it migrates as a single band with a molecular mass of approximately 30 kDa. In the absence of reducing agents, it appears as two bands with apparent molecular weights of ~25 and ~60 kDa, indicating potential disulfide bonding in its native form .

How is PGLYRP1 different from other members of the PGRP family?

PGLYRP1 is one of four mammalian PGRPs, which also include PGRP-L/Tag-L/PGLYRP2, PGRP-Iα/PGLYRP3, and PGRP-Iβ/PGLYRP4. These proteins are named according to the length of their transcripts: "S" for short (PGLYRP1), "L" for long (PGLYRP2), and "I" for intermediate (PGLYRP3 and PGLYRP4) .

While all four PGRPs contain conserved PGRP domains, PGLYRP1 is distinguished by being a secreted protein, whereas the other three members (PGLYRP2, PGLYRP3, and PGLYRP4) are membrane-bound molecules. These membrane-bound PGRPs contain two membrane-spanning segments with both N- and C-termini positioned extracellularly and a joining cytoplasmic domain. In contrast, PGLYRP1 can be secreted from cells and may interact with other components of the innate immunity system through paracrine signaling .

What is the evolutionary conservation of PGLYRP1 across species?

PGLYRP1 is highly conserved across mammalian species. Human PGLYRP1 shares significant amino acid identity with its counterparts in other mammals: 72% with mouse PGLYRP-S, 71% with bovine PGLYRP-S, and 70% with rat PGLYRP-S . This high degree of conservation suggests that PGLYRP1 plays a fundamental role in innate immunity that has been preserved throughout mammalian evolution.

The PGRP family as a whole is conserved even more broadly, from insects to mammals, though insects typically have more PGRP genes (Drosophila has thirteen, while mammals have only four). This conservation across diverse taxonomic groups highlights the essential role of these proteins in innate immune defense mechanisms .

What mechanisms underlie PGLYRP1's antimicrobial activity?

PGLYRP1 exhibits antimicrobial activity through multiple mechanisms:

How does PGLYRP1 contribute to cancer immune evasion mechanisms?

Recent research has identified PGLYRP1 as a novel cancer stem cell (CSC)-associated marker that plays a crucial role in immune evasion, particularly in pancreatic ductal adenocarcinoma (PDAC). The mechanisms involved include:

• Protection against immune-mediated cytotoxicity: PGLYRP1 overexpression protects cancer stem cells from immune-mediated cytotoxic effects, contributing to their survival and persistence .

• Resistance to macrophage phagocytosis: PGLYRP1 confers resistance to macrophage-mediated killing, allowing cancer cells to evade this important immune surveillance mechanism .

• Inhibition of T-cell-mediated killing: Cancer cells with high PGLYRP1 expression show increased resistance to T-cell-mediated killing, further supporting tumor growth .

• TNFα-regulated expression: Mechanistically, tumor necrosis factor alpha (TNFα) regulates PGLYRP1 expression, which then interferes with the immune tumor microenvironment (TME) landscape .

• Promotion of immunosuppression: PGLYRP1 promotes myeloid cell-derived immunosuppression and activated T-cell death, creating a more favorable environment for tumor progression .

These findings establish PGLYRP1 as a potential therapeutic target in cancer, particularly for improving immunotherapy approaches in PDAC treatment.

What is known about PGLYRP1's role in inflammatory diseases?

PGLYRP1 has been associated with several inflammatory conditions:

• Inflammatory bowel disease (IBD): Dysregulation of PGLYRP1 has been implicated in inflammatory bowel disease, suggesting a role in gut inflammation regulation .

• ST-elevation myocardial infarction: PGLYRP1 has been studied in the context of ST-elevation myocardial infarction, indicating potential involvement in cardiovascular inflammation .

• Atherosclerosis: Research has linked PGLYRP1 to atherosclerosis development, possibly through modulation of inflammatory responses in arterial walls .

• Rheumatoid arthritis (RA): PGLYRP1 may contribute to the inflammatory processes in rheumatoid arthritis, an autoimmune disease characterized by joint inflammation .

• Skin melanoma and renal carcinoma: Beyond PDAC, PGLYRP1 has also been studied in relation to skin melanoma and renal carcinoma, suggesting broader implications in cancer biology .

Studies with PGLYRP1-deficient mice have shown increased susceptibility to infections with non-pathogenic bacteria. While neutrophils from these knockout mice exhibit normal phagocytosis of bacteria, they are defective in intracellular killing and digestion of non-pathogenic bacteria, highlighting PGLYRP1's role in neutrophil-mediated bacterial clearance .

How can researchers produce and purify recombinant human PGLYRP1?

Production and purification of recombinant human PGLYRP1 can be approached through the following methodological steps:

• Expression system selection: Recombinant human PGLYRP1 can be produced in eukaryotic expression systems such as CHO-S cells. This approach allows for proper post-translational modifications that may be critical for function .

• Construct design: A common approach is to fuse the cDNA of human PGLYRP1 with a C-terminal epitope tag (such as a triple DED epitope or 6-His tag) to facilitate purification and detection .

• Stable transfection: After transfecting cells with the PGLYRP1 expression construct, stable clones can be generated through positive selection with antibiotics such as G418 (500 μg/ml). The clone with the highest PGLYRP1 production should be selected for protein preparation .

• Protein purification: The recombinant protein can be purified from cultured medium by:

  • Immunoaffinity chromatography using antibodies against the epitope tag

  • Analysis by SDS gel electrophoresis to confirm purity and molecular weight

  • Verification of proper folding through functional assays

• Reconstitution and storage: Commercially available recombinant PGLYRP1 is typically lyophilized from a 0.2 μm filtered solution in PBS with BSA as a carrier protein. It should be reconstituted at approximately 100 μg/mL in sterile PBS and stored to avoid repeated freeze-thaw cycles .

What assays are available for measuring PGLYRP1 binding to bacterial surfaces?

Several assays can be used to assess PGLYRP1 binding to bacterial surfaces:

• Western blot-based binding assay:

  • Incubate PGLYRP1 with bacterial suspensions (either live or fixed with glutaraldehyde)

  • After incubation (typically 1 hour at room temperature), collect bacteria by low-speed centrifugation

  • Wash the bacterial pellets extensively to remove unbound protein

  • Analyze the presence of PGLYRP1 in the pellets by Western blot using antibodies against PGLYRP1 or its epitope tag

  • Compare with input and supernatant fractions to assess binding efficiency

• Stoichiometry determination:

  • Incubate a fixed amount of PGLYRP1 with varying amounts of bacterial cells

  • After washing, analyze both pellets and supernatants for the presence of PGLYRP1

  • This approach allows quantification of how many PGLYRP1 molecules can bind per bacterial cell

• Microscopy-based methods:

  • Fluorescently label PGLYRP1 (directly or via antibodies)

  • Visualize binding to bacterial surfaces using fluorescence microscopy

  • This can provide spatial information about the distribution of PGLYRP1 on bacterial surfaces

• ELISA-based binding assays:

  • Coat plates with whole bacteria or purified bacterial components

  • Detect bound PGLYRP1 using specific antibodies in an ELISA format

  • This approach can be quantitative and high-throughput

How can PGLYRP1 be quantified in biological samples?

For quantification of PGLYRP1 in biological samples, researchers can employ the following methods:

• DuoSet ELISA:

  • Commercial ELISA development kits, such as the Human PGLYRP1/PGRP-S DuoSet ELISA, are available for measuring natural and recombinant human PGLYRP1

  • These kits contain optimized capture and detection antibody pairings with recommended concentrations

  • The standard diluent is suitable for analysis of most cell culture supernatant samples

  • For complex matrices such as serum and plasma, specific diluents should be evaluated prior to use

• Western blotting:

  • PGLYRP1 can be detected in biological samples through Western blotting using specific antibodies

  • This approach is particularly useful when analyzing expression in cell lysates or tissue extracts

  • Molecular weight markers can help confirm the identity of PGLYRP1 bands (~25-30 kDa as monomer, ~60 kDa as dimer)

• Liquid chromatography-mass spectrometry (LC-MS):

  • For more precise quantification, LC-MS methods can be developed

  • This approach is particularly valuable for research on PGLYRP1 as a biomarker in clinical samples

  • In pancreatic cancer studies, secreted PGLYRP1 levels were significantly elevated in patient samples, suggesting utility as a biomarker

• Flow cytometry:

  • For cell-associated PGLYRP1, flow cytometry using fluorescently labeled anti-PGLYRP1 antibodies can provide quantitative data on a per-cell basis

  • This approach is valuable when studying cell-type-specific expression patterns

What experimental models are suitable for studying PGLYRP1 function?

Researchers can employ various experimental models to study PGLYRP1 function:

• Cell culture models:

  • Macrophage cell lines (e.g., murine ANA-1 cells) for studying PGLYRP1's effects on bacterial phagocytosis and killing

  • Human macrophages differentiated from peripheral blood monocytes for confirmation in primary cells

  • Cancer cell lines for studying PGLYRP1's role in tumor immunity

  • Methods for transfection and stable expression of PGLYRP1 have been established in various cell types

• Bacterial infection models:

  • Listeria monocytogenes is a well-established model for studying PGLYRP1's role in intracellular bacterial killing

  • Other bacterial species including E. coli, S. aureus, and S. pneumoniae have been used to study PGLYRP1 binding to bacterial surfaces

• Genetic manipulation approaches:

  • PGLYRP1 knockout mice provide valuable insights into the protein's role in host defense

  • These mice show increased susceptibility to infections with non-pathogenic bacteria

  • Their neutrophils exhibit normal phagocytosis but defective intracellular killing of bacteria

• Cancer models:

  • The KPC mouse model of pancreatic cancer has been used to study PGLYRP1's role in cancer stem cells and immune evasion

  • PGLYRP1 knockout in cancer cells impeded tumor growth in immunocompetent mice

  • These models help elucidate PGLYRP1's role in the tumor microenvironment and potential as a therapeutic target

What is the potential of PGLYRP1 as a biomarker in disease states?

PGLYRP1 shows significant promise as a biomarker in several disease contexts:

• Pancreatic ductal adenocarcinoma (PDAC):

  • Secreted PGLYRP1 levels are significantly elevated in serum samples from patients with PDAC

  • This elevation makes it a potential predictive biomarker that could be measured in liquid biopsy samples

  • PGLYRP1 levels may help stratify patients at early disease stages, potentially improving treatment decisions

• Inflammatory diseases:

  • Based on its known associations with inflammatory bowel disease, atherosclerosis, and rheumatoid arthritis, PGLYRP1 could serve as a biomarker for disease activity or treatment response

  • Quantitative measurements in patient samples could provide insights into disease mechanisms and progression

• Bacterial infections:

  • Given PGLYRP1's role in antibacterial defense, altered levels might indicate specific types of bacterial infections or predict infection outcomes

  • Studies with PGLYRP1-deficient mice suggest its importance in clearing non-pathogenic bacteria, indicating potential utility in monitoring dysbiosis or bacterial translocation

How might PGLYRP1 be targeted therapeutically?

PGLYRP1 presents several potential therapeutic strategies:

• Cancer immunotherapy:

  • Inhibition of PGLYRP1 could reduce cancer stem cell immune evasion, potentially making tumors more susceptible to immune attack

  • PGLYRP1 inhibitors could be developed for combination with existing immunotherapies or immune checkpoint inhibitors

  • This approach appears particularly promising for pancreatic cancer, which has historically been resistant to immunotherapy

• Bacterial infections:

  • Recombinant PGLYRP1 or PGLYRP1-mimetic peptides could potentially enhance macrophage-mediated killing of intracellular bacteria

  • This approach might be valuable for treating infections with facultative intracellular pathogens like Listeria monocytogenes

• Inflammatory conditions:

  • Modulation of PGLYRP1 activity might help regulate inflammatory responses in conditions like inflammatory bowel disease or rheumatoid arthritis

  • The approach (inhibition or enhancement) would depend on whether PGLYRP1 plays a protective or pathogenic role in the specific condition

What are the key unresolved questions in PGLYRP1 research?

Despite significant advances in our understanding of PGLYRP1, several important questions remain:

• Molecular mechanisms: The precise molecular mechanisms by which PGLYRP1 enhances intracellular bacterial killing remain to be fully defined. While effects on oxidative burst and cytokine production have been observed, these appear too late to explain the rapid decrease in intracellular bacterial survival .

• Signaling pathways: How PGLYRP1 interacts with other components of innate immunity through paracrine signaling requires further investigation. The downstream signaling pathways activated by PGLYRP1 in different cell types need to be elucidated.

• Cancer stem cell biology: The mechanisms by which PGLYRP1 becomes overexpressed in cancer stem cells and its role in stemness maintenance versus immune evasion require further clarification .

• Therapeutic targeting: The development of specific inhibitors of PGLYRP1 and their testing in preclinical models will be critical for advancing potential therapeutic applications, particularly in cancer treatment .

• Biomarker validation: Larger clinical studies are needed to validate PGLYRP1 as a biomarker in various disease states, particularly in the context of early detection of pancreatic cancer .