The PAM Knockout Raji Polyclonal Cells are a CRISPR/Cas9-edited polyclonal knockout cell population derived from Raji B lymphocytes, with disruption of the gene encoding peptidylglycine alpha-amidating monooxygenase (PAM). This heterogeneous pool enables loss-of-function studies without clonal selection bias, providing a robust model to investigate C-terminal peptide amidation in B cell biology.
The Raji cell line, established from a Burkitt lymphoma, is an EBV-positive human B lymphoblast model widely used to study immune responses and antibody production. Raji cells express receptors for various neuropeptides, making them suitable for exploring neuro-immune communication and peptide-induced signaling in B lymphocytes.
PAM is a bifunctional enzyme composed of peptidylglycine ??-hydroxylating monooxygenase (PHM) and peptidylglycine ??-amidating lyase (PAL) domains that sequentially convert peptidylglycine substrates into amidated products. This copper-, ascorbate-, and oxygen-dependent reaction is critical for activating numerous neuropeptides and peptide hormones. PAM transcription is suppressed by the RE1-silencing transcription factor (REST) and modulated by intracellular copper, cAMP, and calcium levels. Amidated peptides, including substance P, neuropeptide Y, and oxytocin, bind specific G protein-coupled receptors??such as NK1R, NPY1R, and oxytocin receptor??to initiate downstream cAMP and calcium signaling cascades. PAM also physically interacts with protein disulfide isomerase (PDI) and endoplasmic reticulum-resident chaperones to ensure proper folding.
In Raji B lymphocytes, the loss of PAM abrogates the production of bioactive amidated peptides, thereby disrupting autocrine and paracrine signaling through their cognate GPCRs. This knockout model enables systematic investigation of amidation-dependent processes in immune cells, including effects on B cell proliferation, cytokine secretion, migration, and survival. It also offers a platform to study how neuropeptide maturation influences B cell function and contributes to neuro-immune crosstalk, with implications for understanding the role of B cells in neuroendocrine and inflammatory disorders.
The PAM Knockout Raji Polyclonal Cells are well-suited for diverse research applications, such as high-throughput screens for PAM modulators, mechanistic studies of neuropeptide signaling in B lymphocytes, and exploration of neuro-immune interactions in diseases like neuroendocrine disorders, obesity, diabetes, chronic pain, and psychiatric conditions. Representative assays include Western blotting and RT-qPCR for PAM validation, amidation activity assays, flow cytometry for phenotypic profiling, cytokine secretion analysis, and migration/invasion assays to assess B cell trafficking. Phospho-signaling analysis can further delineate altered intracellular pathways. For additional information, please contact Ascent Research.
