Elsevier

Biophysical Chemistry

Volume 219, December 2016, Pages 1-8
Biophysical Chemistry

Amyloidogenesis of the amylin analogue pramlintide

https://doi.org/10.1016/j.bpc.2016.09.007Get rights and content

Highlights

  • •

    Pramlintide (25,28,29 Pro-human amylin) is an amylinomimetic compound.

  • •

    Pramlintide assembly into oligomers as observed by ESI-IMS-MS

  • •

    Pramlintide was shown to aggregate at mild acidic to neutral solutions.

  • •

    The amyloid nature of pramlintide aggregates were confirmed by TEM, AFM, XRD, FTIR.

  • •

    The results suggest an universal amyloid behavior of amylin analogues.

Abstract

Amylin is a pancreatic peptide hormone co-secreted along with insulin by the β-cells. It is found in amyloid deposits in both type 2 diabetic individuals and elder non-diabetic. The triple proline amylinomimetic compound (25,28,29-Pro-human amylin) named pramlintide was designed aiming to solve the solubility and amyloid characteristics of human amylin. We have found by using ion mobility spectrometry-based mass spectrometry that pramlintide is able to assembly into multimers. Pramlintide formed amyloid fibrils in vitro in a pH-dependent kinetic process within a few hours, as followed by thioflavin T, quantification of soluble peptide and further characterized by transmission electron microscopy, atomic force microscopy and X-ray diffraction. These data indicate that pramlintide can form amyloid fibers.

Introduction

Amylin (also named islet amyloid polypeptide – IAPP) is co-secreted with insulin by the pancreatic β-cell [1], [2], and thus deficient in type 1 diabetic individuals. Amylin was discovered after isolation from amyloid deposits in diabetic cats and type-2 diabetic humans and reported simultaneously by two independent groups [3], [4]. Amylin is a key hormone involved in the regulation of appetite, gastric emptying, glucagonemia, among other physiologic roles [5], [6], and thus it was considered as a potential therapeutic agent in diabetes [7], [8]. However, its immediate therapeutic applicability was hampered due to the limited solubility and amyloid propensity of human amylin.

The correlational observation between proline-rich amylin variants among species and the lack of noticeable amyloid deposit in vivo [9] motivated the development of the triple-proline (25,28,29Pro-human amylin) amylinomimetic hormone denominated pramlintide (Fig. 1), which showed to present improved physico-chemical properties (such as solubility) and similar pharmacologic properties compared to natural amylin [10].

Previous studies provided data with evidence for the propensity for amyloid material of the murine amylin and fragments in vitro [11], [12], [13], and oligomers and birefringent material in vivo in rodents [14], [15]. These results have been considered of minor importance, most likely to resemble non-specific or secondary artifactual measurements when compared to the magnitude of human amylin tinctorial and immunogenic properties. We have also recently shown that murine amylin can assemble into soluble oligomers and mature fibrils in vitro [16], confirmed independently by others groups [13], [17], [18].

Given the propensity of amyloid aggregation of murine amylin thought of decreased tinctorial intensity when compared to the human amylin, an appealing hypothesis is that other proline-rich amylin analogues would be also able to self-assembly into oligomers and amyloid material. Support for this hypothesis comes from evidences of amyloid fibrils of pramlintide at pH 7.5 [12], although barely detectable by the fibril-sensitive fluorescence probe thioflavin T (ThT) up to 20 days at room temperature and high concentration, and thus also considered of only marginal importance. A recent study reported no detectable amyloid aggregation with pramlintide in Tris buffer pH 7.4 [19], despite its known propensity for precipitation at pH above 5.5 [20].

Here we report a systematic evaluation of the physico-chemical properties of pramlintide and its potential to oligomerization and amyloid aggregation. We explored the dependence of pH over a broad range, following evaluation by high definition electron-spray ionization-mass spectroscopy coupled to ion mobility (ESI-IMS-MS), ThT and ultrastructural techniques such as transmission electron microscopy (TEM), atomic force microscopy (AFM), X-ray diffraction and infrared spectroscopy. We further provide evidences that the amyloid aggregation event is hampered under tris buffer, while observable in phosphate buffer solution.

Section snippets

Materials

Carboxy-amidated, C2-C7 disulfide bond, human amylin (TFA salt, lot #85190) and pramlintide (TFA salt, lot #89437; acetate salt, lot #101083) were purchased from Genemed Biotech Inc. (CA, USA). The identity (by ESI-MS) and purity (C-18 reversed phase chromatography) of the peptides were confirmed by quality control certificate provided intra-batch and also by our own measurements by both MALDI-ToF-MS and ESI-MS. The stock peptide solutions were prepared at 10 mg/mL (2.55 mM) in DMSO and stored at

Oligomerization of pramlintide

We have tested whether pramlintide can assemble into oligomers by using electrospray ionization–mass spectrometry coupled to ion mobility spectrometry (ESI-IMS-MS). This technique allows the separation of charge state during migration in a gas-pressurized chamber, resulting in a 2D-pattern of mobility versus mass-charge ratio (m/z), providing visualization of distinct oligomeric state with distinct similar charged state but with overlapping m/z, or even the detection of similar oligomeric state

Discussion

Type 2 diabetes mellitus (T2DM) is considered a chronic degenerative disease in which the rate of apoptosis overcomes the regeneration of β-cells, resulting in a net loss of hormone-secreting cells [32], [33] along with a progressive loss of ability of cosecretion of insulin and amylin (Ludvik et al. 1991). The correlation between these phenomena have been observed in organism in which their natural amylin are not proline-rich variant [9], [34], [35]. Since then, there has been an a priori

Conclusions

We have shown that pramlintide, along with murine amylin [13], [16], can be converted into amyloid material in vitro, giving rise to the hypothesis for a universal amyloid propensity of amylin analogues. However, it remains unclear why these proline-rich variants are less responsive to tinctorial properties and to immunobinding, which shall be addressed in further work. In addition, the design, characterization and validation of bioanalytical methods to detect oligomeric and amyloid material in

Conflict of interest

Prof. Luis Mauricio T. R. Lima, is applicant of patents regarding controlled release of pancreatic hormones

Acknowledgments

We would like to thank MSc Daniela Lourenço, Dr. Soraya Ochs and Ivone Rosa (DIMAV-INMETRO) for excellent technical services, Prof. Jackson Menezes (IQ/IF-UFF) for providing access to the diffraction facility and Dr. Luciano B. Candido for technical support at the LNLS-MX2 beamline. This research was supported by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Fundação de Amparo à Pesquisa do Estado

References (42)

  • H. LeVine

    Quantification of beta-sheet amyloid fibril structures with thioflavin T

    Methods Enzymol.

    (1999)
  • R. Sabaté et al.

    On the binding of thioflavin-T to HET-s amyloid fibrils assembled at pH 2

    J. Struct. Biol.

    (2008)
  • M.E. Oskarsson et al.

    In vivo seeding and cross-seeding of localized amyloidosis: a molecular link between type 2 diabetes and Alzheimer disease

    Am. J. Pathol.

    (2015)
  • S.E. Kahn et al.

    Evidence of cosecretion of islet amyloid polypeptide and insulin by beta-cells

    Diabetes

    (1990)
  • G.J. Cooper et al.

    Purification and characterization of a peptide from amyloid-rich pancreases of type 2 diabetic patients

    Proc. Natl. Acad. Sci.

    (1987)
  • P. Westermark et al.

    Amyloid fibrils in human insulinoma and islets of Langerhans of the diabetic cat are derived from a neuropeptide-like protein also present in normal islet cells

    Proc. Natl. Acad. Sci.

    (1987)
  • A.L. Castle et al.

    Amylin influences insulin-stimulated glucose metabolism by two independent mechanisms

    Am. J. Phys.

    (1998)
  • S.E. Inzucchi et al.

    Management of hyperglycemia in type 2 diabetes, 2015: a patient-centered approach: update to a position statement of the American Diabetes Association and the European Association for the Study of diabetes

    Diabetes Care

    (2015)
  • P. Westermark et al.

    Islet amyloid polypeptide: pinpointing amino acid residues linked to amyloid fibril formation

    Proc. Natl. Acad. Sci. U. S. A.

    (1990)
  • A.A. Young et al.

    Preclinical pharmacology of pramlintide in the rat: comparisons with human and rat amylin

    Drug Dev. Res.

    (1996)
  • S.B. Chargé et al.

    Effect of pH and insulin on fibrillogenesis of islet amyloid polypeptide in vitro

    Biochemistry (Mosc)

    (1995)

    • Cited by (35)

    • Amyloidogenicity of peptides targeting diabetes and obesity

      2022, Colloids and Surfaces B: Biointerfaces

    • Amyloidogenesis of feline amylin and plasma levels in cats with diabetes mellitus or pancreatitis

      2021, Domestic Animal Endocrinology
      Citation Excerpt :

      However, no comparative assays have been reported to date. In fact, the triple proline analogues rodent amylin and the triple-proline (25,28,29Pro) human amylin analogue pramlintide have long been assumed to be nonamyloidogenic and nontoxic in vitro and in vivo, until recent works revealed their propensity in forming amyloid material [26–30]. In the present study, we investigated the amyloid nature of cat amylin compared with other species, exploring the aggregation pattern of cat, human, and murine amylin as well as the amylin analogue pramlintide in vitro.

    • Dietary zinc restriction promotes degeneration of the endocrine pancreas in mice

      2020, Biochimica et Biophysica Acta - Molecular Basis of Disease
      Citation Excerpt :

      Human amylin forms amyloid fibrils [27,28], but the aggregation of the murine variant is still under discussion. Previously, our group demonstrated that murine amylin and the amylin analogue pramlintide can aggregate in vitro [29–31]. Toxic oligomers in pancreatic islets can be found in nontransgenic mice [32].

    • Amylin and amylin receptors in Alzheimer’s disease

      2020, Genetics, Neurology, Behavior, and Diet in Dementia: The Neuroscience of Dementia, Volume 2

    • BZ043, a novel long-acting amylin analog, reduces gastric emptying, food intake, glycemia and insulin requirement in streptozotocin-induced diabetic rats

      2019, Peptides

    • Physico-chemical properties of co-formulated fast-acting insulin with pramlintide

      2018, International Journal of Pharmaceutics
      Citation Excerpt :

      An amyloid aggregation assay was conducted using 50 μM each polypeptide in 50 mM buffer pH 7.0 or pH 5.0, and ThT as amyloid fluorescent probe. Pramlintide showed a time-dependent increasing ThT fluorescence compatible with the formation of amyloid material at pH 7.0 (da Silva et al., 2016) (Supp. Fig. S1A), being more prominent in sodium phosphate (Fig. 5A) than in ammonium acetate (Fig. 5B) or sodium acetate (Fig. 5C) solutions. Instead, pramlintide showed no detectable increase in ThT fluorescence in these buffers at pH 5.0 (Fig. 5D, E and F), confirming its enhanced stability at acidic milieu regardless of the buffer agent.

    View all citing articles on Scopus
    View full text
    © 2016 Elsevier B.V. All rights reserved.