Peptide-Peptide Co-Assembly: A Design Strategy for Functional Detection of C-peptide, A Biomarker of Diabetic Neuropathy

Abstract

Diabetes-related neuropathy is a debilitating condition that may be averted if it can be detected early. One possible way this can be achieved at low cost is to utilise peptides to detect C-peptide, a biomarker of diabetic neuropathy. This depends on peptide-peptide co-assembly, which is currently in a nascent stage of intense study. Instead, we propose a bead-based triple-overlay combinatorial strategy that can preserve inter-residue information during the screening process for a suitable complementary peptide to co-assemble with C-peptide. The screening process commenced with a pentapeptide general library, which revealed histidine to be an essential residue. Further screening with seven tetrapeptide focused libraries led to a table of self-consistent peptide sequences that included tryptophan and lysine at high frequencies. Three complementary nonapeptides (9mer com-peptides), wpkkhfwgq (Trp-D), kwkkhfwgq (Lys-D), and KWKKHFWGQ (Lys-L) (as a negative control) were picked from this table for co-assembly studies with C-peptide. Attenuated total reflectance Fourier transform infrared (ATR-FTIR) and circular dichroism (CD) spectroscopies were utilized to study inter-peptide interactions and changes in secondary structures respectively. ATR-FTIR studies showed that there is indeed inter-peptide interaction between C-peptide and the tryptophan residues of the 9mer com-peptides. CD studies of unaggregated and colloidal C-peptide with the 9mer com-peptides suggest that the extent of co-assembly of C-peptide with Trp-D is greatest, followed by Lys-D and Lys-L. These results are promising and indicate that the presented strategy is viable for designing and evaluating longer complementary peptides, as well as complementary peptides for co-assembly with other polypeptides of interest and importance. We discuss the possibility of designing complementary peptides to inhibit toxic amyloidosis with this approach.

Keywords: C-peptide; amyloidosis; biomarker; co-assembly; design; detection; diabetic neuropathy; inhibition; peptide-peptide.

Figure 1

The various coloured circles represent different amino acid residues. The dotted arrows point to the segments of pentapeptide sequences that can be picked up a focussed library possessing those segments. (A) Illustration of how inter-peptide interaction is also affected by intra-peptide residue interaction. In this example, residue 3 of C-peptide may interact favourably with residue 1 of the com-peptide. This would affect how residues 2–5 of the com-peptide interact with residues 4–8 of the C-peptide. (B) Illustration of the triple-overlay strategy for the search of a complementary peptide (com-peptide) to co-assemble with C-peptide. The pentapeptide in Line 2 can pick up two further pentapeptide sequences in Lines 3 and 4 via the consensus triplet sectors.

Figure 2

Summary of the bead screening results of a 5mer general library against C-peptide. Histidine, the aromatic residues (phenylalanine, tyrosine, and tryptophan), and the remaining residues are respectively highlighted in red, blue, and grey. The numbers indicate the number of peptide sequences successfully sequenced. The histogram on the left represents the composite results of the four separate rounds of screening (on the right) with the 5mer general library. The histograms are prepared with the publicly available WebLogo program [30]. These results show that it is consistently favourable for His to be in position 3.

Figure 3

Summary of the screening results of the three His-centric focused libraries against C-peptide (panels 1–3). The twin blue bars in positions 5 (row 1), 3 (row 2), and 1 (row 3) correspond to His. The results of row 1 indicate that it is favourable for Trp to be in positions 1–3 when His is in position 5. The results of row 2 indicate that it is favourable for Lys to be in positions 4 and 5 when His is in position 3. The results of row 3 indicate that it is strongly favourable for Trp to be in positions 5 when His is in position 1.

Figure 4

Summary of the screening results of the four Phe/Tyr/Trp-centric focused libraries against C-peptide (panels 1–4). The red boxes correspond to the position of the focused library in which variation in residues is limited. The green arrows highlight the high frequency at which Trp appeared in the peptide sequences that interacted favourably with C-peptide during the screening process.

Figure 5

Illustration of the process of rejecting and accepting complementary peptide sequences to C-peptide. In the left table, the pink and red libraries, as well as the light and dark blue libraries, possess kw (positions 3 and 4) in each coloured library and in two different rounds (e.g., pink and red), so they fulfil the two criteria for acceptance. However, positions 6 and 7 fail one or both criteria (lower oval). For example, kf may appear in two different rounds (Criterion 1), but it only appears once in each round. Both fg and yk only appear once in either round. Thus, even though fg fulfils both criteria in the orange/dark yellow/cream library (upper oval), this whole table is rejected because positions 1–5 cannot link up with positions 5–9. On the other hand, all sectors in the right table fulfil both criteria of acceptance, so the self-consistent table on the right is accepted.

Figure 6

ATR-FTIR spectra of C-peptide, Trp-D, Lys-D, and Lys-L. Panel (A) compares and contrasts the IR spectra of C-peptide, 9mer com-peptides, and the mixtures. Panels (B–D) compare the experimental IR spectra of C-peptide+com-peptide with the theoretical spectrum, C-peptide and the respective com-peptide. The arrows in spectra (B–D) indicate the main difference between the experimental and theoretical spectra. The disappearance of the peaks around 1200 cm⁻¹ suggests that there is interaction of Trp with C-peptide during co-assembly. The bump around 3300 cm⁻¹ is due to imperfect subtraction of the background water stretching mode.

Figure 7

CD spectral time course of (A) C-peptide, (B) Trp-D, (C) Lys-D, and (D) Lys-L. The inset in spectrum A shows the change in C-peptide over time–(left) clear at the start, (right) colloidal after 53 days. The CD spectra show that C-peptide changes slowly over time, but the three 9mer com-peptides remain essentially unchanged over the same period.

Figure 8

CD spectral time course of unaggregated C-peptide co-assembled with (A) Trp-D, (B) Lys-D, and (C) Lys-L respectively. The relative sizes of the vertical arrows denote the relative change in ellipticity at 198 nm. Panel (D) illustrates the change in the mean residue ellipticity at 198 nm relative to the simulated spectra of non-interaction between C-peptide and the 9mer com-peptides; the line due to C-peptide reflects changes relative to 0 day. It shows that Trp-D interacts to the greatest extent with C-peptide, followed by Lys-D and (minimally) Lys-L. It also shows how the electrolyte-like 9mer com-peptides stopped the aggregation of C-peptide.

Figure 9

CD spectral time course of colloidal C-peptide co-assembled with (A) Trp-D, (B) Lys-D, and (C) Lys-L respectively. The relative sizes of the vertical arrows denote the relative change in ellipticity at 198 nm. Panel (D) illustrates the change in the mean residue ellipticity at 198 nm relative to the simulated spectra of non-interaction between colloidal C-peptide and the 9mer com-peptides; the line due to C-peptide reflects changes relative to 0 day. It shows that Lys-L interacts to the greatest extent with colloidal C-peptide (biggest decrease in rate of increase of [$\theta$]₁₉₈ relative to that of C-peptide), followed by Lys-D and Trp-D.

Figure 10

Job plots of C-peptide and (A) Trp-D, (B) Lys-D, and (C) Lys-L at a total concentration of 1 mM as determined at 198 nm. The standard deviations are derived from three separate measurements over the course of two weeks. The absence of a well-defined peak in the plots suggests that the co-assembly of C-peptide and the 9mer com-peptides is not optimized yet.

Scheme 1

Reaction for the labeling of C-peptide for visualization via fluorescence during the bead sorting process.

Scheme 2

Illustration of the bead screening process for a complementary peptide that can co-assemble with C-peptide. The process begins with incubating fluorescein-labelled C-peptide with the bead library. Sequences that can co-assemble with C-peptide will fluoresce on excitation with a blue laser, which will be sorted and collected in a 96-microwell. The collected beads are then treated with cyanogen bromide to cleave the peptides off the beads. Finally, the recovered peptides are analysed by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) and sequenced by the PEAKS mass spectrometry program.