E-peptides control bioavailability of IGF-1

Abstract

Insulin-like growth factor 1 (IGF-1) is a potent cytoprotective growth factor that has attracted considerable attention as a promising therapeutic agent. Transgenic over-expression of IGF-1 propeptides facilitates protection and repair in a broad range of tissues, although transgenic mice over-expressing IGF-1 propeptides display little or no increase in IGF-1 serum levels, even with high levels of transgene expression. IGF-1 propeptides are encoded by multiple alternatively spliced transcripts including C-terminal extension (E) peptides, which are highly positively charged. In the present study, we use decellularized mouse tissue to show that the E-peptides facilitate in vitro binding of murine IGF-1 to the extracellular matrix (ECM) with varying affinities. This property is independent of IGF-1, since proteins consisting of the E-peptides fused to relaxin, a related member of the insulin superfamily, bound equally avidly to decellularized ECM. Thus, the E-peptides control IGF-1 bioavailability by preventing systemic circulation, offering a potentially powerful way to tether IGF-1 and other therapeutic proteins to the site of synthesis and/or administration.

Figure 1. Structure of the rodent IGF-1 gene.

Exons 1 and 2 are transcribed from different promoters. Differential splicing gives rise to two different signal peptides (SP1 and SP2), which include a common C-terminal sequence encoded by Exon 3. Exon 3 also encodes the N-terminal part of the mature IGF-1 B chain. Exon 4 encodes the remaining mature IGF-1 protein (B,C,A and D chains), and also encodes the common N-terminal sequence of the E-peptides. Differential splicing excluding Exon 5 gives rise to the IGF-1Ea propeptide, or a longer IGF-1Eb propeptide when Exon 5 is included. Protease cleavage (arrowheads) removes the E peptides to produce the mature IGF-1 protein.

Figure 2. IGF-1 expression and secretion in transgenic animals.

A) Western blot analysis of IGF-1 transgene levels in quadriceps muscle of 3 months old male mice. B) Total IGF-1 levels in the blood serum of 3 months old transgenic male mice compared to WT littermates as determined by ELISA.

Figure 3. E-peptides promote binding of IGF-1 to negatively charged surfaces.

A) Growth medium (10 uL) from transiently transfected HEK 293 cells (IGF-1 levels normalised to 200 ng/mL). B) Binding of IGF-1 propeptides to positively (amine) (lanes 2–4) and negatively (carboxyl) (lanes 6–8) charged tissue culture plates. The control lane (9) is a mixture of growth media from IGF-1-stop and IGF-1EbCD transfected cells.

Figure 4. E-peptides bind heparin-agarose.

Binding of IGF-1 isoforms to heparin coated agarose beads (lanes 2–4) and control agarose beads (lanes 6–8). The control lane (9) is the growth medium from IGF-1EbCD transfected cells.

Figure 5. Preparation of decellularized tissue as ECM substrate.

Sections of paraffin imbedded control (non-decellularized) skeletal muscle and lung tissue (A-D) or decellularized skeletal muscle and lung tissue (E-H). The sections were stained with hematoxylin/eosin (H/E) or DAPI as indicated.

Figure 6. IGF-1 propeptides bind to the ECM.

Western blot analysis of IGF-1 binding. Lanes 1–4: growth media from HEK 293 cells transfected with IGF-1 expression plasmids encoding either the mature peptide (lane 2) or cleavage deficient IGF-1 propeptides (lanes 3 and 4). Lanes 5–8: same growth media after incubation with decellularizsed tissue. Lanes 9–12: IGF-1 binding to decellularized lung tissue. Lanes 13–16: IGF-1 binding to decellularizsed skeletal muscle tissue.

Figure 7. IGF-1 propeptides bind to the ECM at particular loci.

A–D) Decellularized lung tissue was sectioned and incubated with growth media from HEK 293 cells transfected with IGF-1 expression plasmids (see Materials and Methods for details). Bound IGF-1 was visualized by immunostaining using anti-IGF-1 antibody. E) Quantification of the number of IGF-1 loci. Data is presented as mean (SE) for 20 biological replicates. Two stars corresponds to P<0.01, three stars correspond to P<0.001.

Figure 8. E-peptide mediated binding to the ECM is independent of IGF-1.

A) Schematic representation of the three relaxin based constructs used for the experiments. Fusions of murine relaxin (RLN1)to murine Ea and Eb peptides were produced. The constructs were tagged with biochemical tags (V5 and poly-His). B) V5 Western blot analysis. Lanes 1–4: growth media from HEK 293 cells transfected with RLN1 expression plasmids. Lanes 5–8: same growth media after incubation with decellularizsed tissue. Lanes 9–12: binding of RLN1 constructs to decellularized lung tissue. Lanes 13–16: binding of RLN1 constructs decellularizsed skeletal muscle tissue.