Abstract
The α-hemoglobin-derived dodecapeptide RVD-hemopressin (RVDPVNFKLLSH) has been proposed to be an endogenous agonist for the cannabinoid receptor type 1 (CB(1)). To study this peptide, we have raised mAbs against its C-terminal part. Using an immunoaffinity mass spectrometry approach, a whole family of N-terminally extended peptides in addition to RVD-Hpα were identified in rodent brain extracts and human and mouse plasma. We designated these peptides Pepcan-12 (RVDPVNFKLLSH) to Pepcan-23 (SALSDLHAHKLRVDPVNFKLLSH), referring to peptide length. The most abundant Pepcans found in the brain were tested for CB(1) receptor binding. In the classical radioligand displacement assay, Pepcan-12 was the most efficacious ligand but only partially displaced both [(3)H]CP55,940 and [(3)H]WIN55,212-2. The data were fitted with the allosteric ternary complex model, revealing a cooperativity factor value α < 1, thus indicating a negative allosteric modulation. Dissociation kinetic studies of [(3)H]CP55,940 in the absence and presence of Pepcan-12 confirmed these results by showing increased dissociation rate constants induced by Pepcan-12. A fluorescently labeled Pepcan-12 analog was synthesized to investigate the binding to CB(1) receptors. Competition binding studies revealed K(i) values of several Pepcans in the nanomolar range. Accordingly, using competitive ELISA, we found low nanomolar concentrations of Pepcans in human plasma and ∼100 pmol/g in mouse brain. Surprisingly, Pepcan-12 exhibited potent negative allosteric modulation of the orthosteric agonist-induced cAMP accumulation, [(35)S]GTPγS binding, and CB(1) receptor internalization. Pepcans are the first endogenous allosteric modulators identified for CB(1) receptors. Given their abundance in the brain, Pepcans could play an important physiological role in modulating endocannabinoid signaling.
Highlights
The ␣-hemoglobin-derived peptide RVDPVNFKLLSH was found to interact with cannabinoid cannabinoid receptor type 1 (CB1) receptors
8.02 Ϯ 0.02 7.33 Ϯ 0.12 6.33 Ϯ 0.07 6.84 Ϯ 0.02 a Neoepitopes contain the charge of the C terminus as an essential part of their epitope. monoclonal antibodies (mAbs) 7A5 and 8D4 recognized RVD-Hp␣ with a free C terminus but not the carboxamide derivative (Fig. 1). b pIC50 values were estimated by nonlinear regression analysis of the synthetic peptide RVDPVNFKLLSH competing for the binding of the mAb toward the corresponding ovalbumin-peptide conjugate (Table 1) in Competitive Enzyme-linked Immunosorbent Assay (C-ELISA) under optimized conditions (2C11 (2 ng/ml), 7A5 (10 ng/ml), 8D4 (50 ng/ml); for experiments with 2C11 and 7A5, 50 l of 15 ng/ml ovalbumin-peptide conjugate were coated, and for 8D4, 50 l of 150 ng/ml were used)
The low molecular weight band seen in the mouse brain extracts was significantly reduced already at a concentration of 3 nM RVD-Hp␣, and the band intensity was fully displaced by competitor concentrations above 80 nM (Fig. 2C)
Summary
The ␣-hemoglobin-derived peptide RVDPVNFKLLSH was found to interact with cannabinoid CB1 receptors. The ␣-hemoglobin-derived dodecapeptide RVD-hemopressin (RVDPVNFKLLSH) has been proposed to be an endogenous agonist for the cannabinoid receptor type 1 (CB1). To study this peptide, we have raised mAbs against its C-terminal part. Using an immunoaffinity mass spectrometry approach, a whole family of N-terminally extended peptides in addition to RVD-Hp␣ were identified in rodent brain extracts and human and mouse plasma. We designated these peptides Pepcan-12 (RVDPVNFKLLSH) to Pepcan-23 (SALSDLHAHKLRVDPVNFKLLSH), referring to peptide length. Given their abundance in the brain, Pepcans could play an important physiological role in modulating endocannabinoid signaling
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