Dimerization of a PACAP peptide analogue in DMSO via asparagine and aspartic acid residues

Severs, Joanne C.; Froland, Wayne A.

doi:10.1002/jps.21116

Cited by 10 publications

(7 citation statements)

References 19 publications

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“…Moreover, once a protein is introduced to a human subject, that is, to the blood at pH 7.4, succinimide will rapidly hydrolyze into Asp and isoAsp . Succinimide formation, together with Asn deamidation and Asp isomerization, introduces heterogeneity into proteins and may lead to a number of issues including alterations in structure, intermolecular cross‐linking, aggregation, loss of activity, and even immunogenicity, thereby affecting both efficacy and toxicity …”

Section: Introductionmentioning

confidence: 99%

“…[49][50][51][52] Moreover, once a protein is introduced to a human subject, that is, to the blood at pH 7.4, succinimide will rapidly hydrolyze into Asp and isoAsp. 40,53 Succinimide formation, together with Asn deamidation and Asp isomerization, introduces heterogeneity into proteins and may lead to a number of issues including alterations in structure, 9,54 intermolecular cross-linking, [55][56][57][58][59] aggregation, 60 loss of activity, 48,61,62 and even immunogenicity, 63,64 thereby affecting both efficacy and toxicity. 36,42,65 Detection methods for succinimide are limited both in number and practicality, mainly because of its intrinsic instability, that is, it is rapidly hydrolyzed during tryptic digestion typically carried out at or above neutral pH.…”

Section: Introductionmentioning

confidence: 99%

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Detection and Quantitation of Succinimide in Intact Protein via Hydrazine Trapping and Chemical Derivatization

Klaene

Alfaro

et al. 2014

Journal of Pharmaceutical Sciences

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Formation of aspartyl succinimide (Asu) is a common post-translational modification (PTM) of protein pharmaceuticals under acidic conditions. We present a method to detect and quantitate succinimide in intact protein via hydrazine trapping and chemical derivatization. Succinimide, which is labile under typical analytical conditions, is first trapped with hydrazine to form stable hydrazide and can be directly analyzed by mass spectrometry. The resulting aspartyl hydrazide can be selectively derivatized by various tags, such as fluorescent rhodamine sulfonyl chloride that absorbs strongly in the visible region (570 nm). Our tagging strategy allows the labeled protein to be analyzed by orthogonal methods, including HPLC-UV, LC-MS, and SDS-PAGE coupled with fluorescence imaging. A unique advantage of our method is that variants containing succinimide, after derivatization, can be readily resolved via either affinity enrichment or chromatographic separation. This allows further investigation of individual factors in a complex protein mixture that affect succinimide formation. Some additional advantages imparted by fluorescence labeling include, the facile detection of the intact protein without proteolytic digestion to peptides; and high sensitivity, e.g. without optimization 0.41% succinimide was readily detected. As such, our method should be useful for rapid screening, optimization of formulation conditions and related processes relevant to protein pharmaceuticals.

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Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Detection and Quantitation of Succinimide in Intact Protein via Hydrazine Trapping and Chemical Derivatization

Klaene

Alfaro

et al. 2014

Journal of Pharmaceutical Sciences

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“…As such, mutation of N1251 to either alanine, or one product of asparagine deamidation, aspartic acid, almost completely abrogated antibody binding. This is highly significant since glutamine–glutamate modification has been shown to regulate receptor multimerization, putatively by reducing electrostatic repulsion between helices [33], and similar mechanisms have been proposed for asparagine–aspartate conversion including the regulation of peptide hormone dimerization [34] and activation of extracellular matrix molecules [35]. Moreover, the deamidation of specific asparagines to the exclusion of neighboring asparagines has been shown to regulate protein structure and function [36].…”

Section: Discussionmentioning

confidence: 99%

Modification of the L1-CAM carboxy-terminus in pancreatic adenocarcinoma cells

et al. 2010

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The neural cell adhesion molecule L1 has recently been shown to be expressed in pancreatic adenocarcinoma (PDAC) cells. In this report, we demonstrate that L1 is expressed by moderately- to poorly-differentiated PDAC cells in situ, and that L1 expression is a predictor of poor patient survival. In vitro, reduced reactivity of an anti-L1 carboxy-terminus-specific antibody was observed in the more poorly differentiated fast-growing (FG) variant of the COLO357 population, versus its well-differentiated slow-growing (SG) counterpart, even though they express equivalent total L1. The carboxy-terminus of L1 mediates binding to the MAP kinase-regulating protein RanBPM and mutation of T1247/S1248 within this region attenuates the expression of malignancy associated proteins and L1-induced tumorigenicity in mice. Therefore, we reasoned that the differential epitope exposure observed might be indicative of modifications responsible for regulating these events. However, epitope mapping demonstrated that the major determinant of binding was actually N1251; mutation of T1247 and S1248, alone or together, had little effect on C20 binding. Moreover, cluster assays using CD25 ectodomain/L1 cytoplasmic domain chimeras demonstrated the N1251-dependent, RanBPM-independent stimulation of erk phosphorylation in these cells. Reactivity of this antibody also reflects the differential exposure of extracellular epitopes in these COLO357 sublines, consistent with the previous demonstration of L1 ectodomain conformation modulation by intracellular modifications. These data further support a central role for L1 in PDAC, and define a specific role for carboxy-terminal residues including N1251 in the regulation of L1 activity in PDAC cells.

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“…[81] Secondly, studies on mice with daily administration of PACAP have shown that though PACAP decreases circulating glucose levels, it also stimulates glucagon secretion. Firstly, formulation studies on R3P66, a PACAP analogue, indicated that R3P66 has a short half-life and forms an undesirable dimer that is not useful as a therapeutic agent.…”

Section: Vasoactive Intestinal Peptide (Vip) and Pituitary Adenylate mentioning

confidence: 99%

Structural Aspects of Gut Peptides with Therapeutic Potential for Type 2 Diabetes

Hewage

Venneti

2013

ChemMedChem

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Gut hormones represent a niche subset of pharmacologically active agents that are rapidly gaining importance in medicine. Due to their exceptional specificity for their receptors, these hormones along with their analogues have attracted considerable pharmaceutical interest for the treatment of human disorders including type 2 diabetes. With the recent advances in the structural biology, a significant amount of structural information for these hormones is now available. This Minireview presents an overview of the structural aspects of these hormones, which have roles in physiological processes such as insulin secretion, as well as a discussion on the relevant structural modifications used to improve these hormones for the treatment of type 2 diabetes.

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Dimerization of a PACAP peptide analogue in DMSO via asparagine and aspartic acid residues

Cited by 10 publications

References 19 publications

Detection and Quantitation of Succinimide in Intact Protein via Hydrazine Trapping and Chemical Derivatization

Detection and Quantitation of Succinimide in Intact Protein via Hydrazine Trapping and Chemical Derivatization

Modification of the L1-CAM carboxy-terminus in pancreatic adenocarcinoma cells

Structural Aspects of Gut Peptides with Therapeutic Potential for Type 2 Diabetes

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