ABSTRACT
Amylin is a pancreatic hormone co-secreted with insulin. Human amylin has been shown to form dimers and exhibit high propensity for amyloid fibril formation. We observed the ability of the water-soluble murine amylin to aggregate in water resulting in an insoluble material with Thioflavin T binding properties. Infrared spectroscopy analysis revealed beta-sheet components in the aggregated murine amylin. Morphological analysis by transmission electron microscopy and atomic force microscopy provided access to the fibril nature of the murine amylin aggregate which is similar to amyloid fibrils from human amylin. X-ray diffraction of the murine amylin fibrils showed peaks at 4.7Å and 10Å, a fingerprint for amyloid fibrils. Electron spray ionization-ion mobility spectroscopy-mass spectrometry (ESI-IMS-MS) analysis and crosslinking assays revealed self-association intermediates of murine amylin into high order oligomeric assemblies. These data demonstrate the stepwise association mechanism of murine amylin into stable oligomers, which ultimately converges to its organization into amyloid fibrils.
Subject(s)
Amyloid/metabolism , Islet Amyloid Polypeptide/metabolism , Amyloid/chemistry , Animals , Benzothiazoles , Humans , Islet Amyloid Polypeptide/chemistry , Mice , Microscopy, Atomic Force , Microscopy, Electron, Transmission , Polymerization , Protein Binding , Protein Structure, Secondary , Solutions/chemistry , Spectrometry, Mass, Electrospray Ionization , Spectrophotometry, Infrared , Thiazoles/chemistry , Thiazoles/metabolismABSTRACT
Amylin is a pancreatic hormone that plays important roles in overall metabolism and in glucose homeostasis. The therapeutic restoration of postprandial and basal amylin levels is highly desirable for patients with diabetes who need to avoid glucose excursions. Protein conjugation with polyethylene glycol (PEG) has long been known to be a convenient approach for extending the biological effects of biopharmaceuticals. We have investigated the reactivity of amylin with methoxy polyethylene glycol succinimidyl carbonate and methoxy polyethylene glycol succinimidyl propionate, which have an average molecular weight of 5 kDa. The reaction, which was conducted in both aqueous and organic (dimethyl sulfoxide) solvents, occurred within a few minutes and resulted in at least four detectable products with distinct kinetic phases. These results suggest a kinetic selectivity for PEGylation by succinimidyl derivatives; these derivatives exhibit enhanced reactivity with primary amine groups, as indicated by an evaluation of the remaining amino groups using fluorescamine. The analysis of tryptic fragments from mono- and diPEGylated amylin revealed that conjugation occurred within the 1-11 amino acid region, most likely at the two amine groups of Lys(1). The reaction products were efficiently separated by C-18 reversed phase chromatography. Binding assays confirmed the ability of mono- and diPEGylated amylin to interact with the amylin co-receptor receptor activity-modifying protein 2. Subcutaneous administration in mice revealed the effectiveness of monoPEG-amylin and diPEG-amylin in reducing glycemia; both compounds exhibited prolonged action compared to unmodified amylin. These features suggest the potential use of PEGylated amylin to restore basal amylin levels.
Subject(s)
Islet Amyloid Polypeptide/chemistry , Polyethylene Glycols/chemistry , Amino Acid Sequence , Electrophoresis, Polyacrylamide Gel , Humans , Kinetics , Models, Molecular , Molecular Sequence Data , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Trypsin/chemistryABSTRACT
We modified amylin chemically by conjugating methoxyl polyethyleneglycol succinimidyl carbonate (mPEGSC) of varying molecular weights (1 kDa, 2 kDa and 5 kDa). The reaction occurred within a few minutes, resulting in at least four distinct PEGylated products. The reaction products were separated by reversed-phase chromatography and identified by mass-spectrometry. The monoPEGylated and diPEGylated amylin products were generated rapidly through conjugation to the two amino groups of the N-terminal lysine residue. Both PEGylated amylin products bound to the receptor activity-modifying protein 1 (RAMP1). Pharmacological evaluation by subcutaneous administration in mice of monoPEGylated and diPEGylated amylin obtained with mPEG-SC 5 kDa revealed that both compounds modulated glycemia for longer times than unmodified amylin. Collectively, these data demonstrate the potential of bioconjugation with mPEG for the design of amylin therapeutics with sustained action.
