High efficacy and minimal peptide required for the anti-angiogenic and anti-hepatocarcinoma activities of plasminogen K5

Abstract

Kringle 5(K5) is the fifth kringle domain of human plasminogen and its anti-angiogenic activity is more potent than angiostatin that includes the first four kringle fragment of plasminogen. Our recent study demonstrated that K5 suppressed hepatocarcinoma growth by anti-angiogenesis. To find high efficacy and minimal peptide sequence required for the anti-angiogenic and anti-tumour activities of K5, two deletion mutants of K5 were generated. The amino acid residues outside kringle domain of intact K5 (Pro452-Ala542) were deleted to form K5mut1(Cys462-Cys541). The residue Cys462 was deleted again to form K5mut2(Met463-Cys541). K5mut1 specifically inhibited proliferation, migration and induced apoptosis of endothelial cells, with an apparent two-fold enhanced activity than K5. Intraperitoneal injection of K5mut1 resulted in more potent tumour growth inhibition and microvessel density reduction than K5 both in HepA-grafted and Bel7402-xenografted hepatocarcinoma mouse models. These results suggested that K5mut1 has more potent anti-angiogenic activity than intact K5. K5mut2, which lacks only the amino terminal cysteine of K5mut1, completely lost the activity, suggesting that the kringle domain is essential for the activity of K5. The activity was enhanced to K5mut1 level when five acidic amino acids of K5 in NH(2) terminal outside kringle domain were replaced by five serine residues (K5mut3). The shielding effect of acidic amino acids may explain why K5mut1 has higher activity. K5, K5mut1 and K5mut3 held characteristic β-sheet spectrum while K5mut2 adopted random coil structure. These results suggest that K5mut1 with high efficacy is the minimal active peptide sequence of K5 and may have therapeutic potential in liver cancer.

Fig 1

Schematic structure and generation of K5 and its deletion mutants. (A) The primary sequence of K5 and its mutants. (B) The schematic structure of K5 and its mutants. Two deletion mutants were designed according to the structure and disulfide bond distribution of K5 (Pro452-Ala542). K5mut1 (Cys462-Cys541) retained the intact kringle domain and deleted the amino acid residues of both terminals outside kringle domain of K5. K5mut2 (Met463-Cys541) opened the first disulfide bond by deleting the amino acids of Cys462 on the basis of K5mut1 structure. (C) The production and identification of recombinant K5 and its mutant proteins. The top panel is SDS-PAGE with Coomassie blue staining while the bottom panel is Western blot analysis with antibody specific to His-tag; Marker: Protein marker; Lane1: Purified recombinant protein of K5; Lane2: Purified recombinant protein of K5mut1; Lane3: Purified recombinant protein of K5mut2.

Fig 2

Recombinant K5 and mutants possess consistent disulfide numbers and folding with the native peptides by orthogonal digestion combining with MALDI-Q TOF mass spectrometry analysis. (A) Expected peptide fragments generated for K5 protein digested with trypsin followed by Asp-N endopeptidase treatment under non-reducing condition (top). Spectra confirming Cys483:524, Cys462:541 and Cys512:536 disulfide bond under non-reducing (bottom left) and reducing conditions (bottom right). (B) Expected peptide fragments generated for K5mut1 protein digested with trypsin followed by Asp-N endopeptidase treatment under non-reducing condition (top). Spectra confirming Cys483:524, Cys462:541 and Cys512:536 disulfide bond under non-reducing (bottom left) and reducing conditions (bottom right). (C) Expected peptide fragments generated for K5mut2 protein digested with trypsin alone under non-reducing condition (top). Spectra confirming the disulfide bond of Cys483:524 and Cys512:536 under non-reducing (bottom left) and reducing conditions (bottom right).

Fig 3

Effects of K5 and its deletion mutants on endothelial cell proliferation, apoptosis, migration and other cells proliferation. Cells were treated with the recombinant K5, K5mut1 and K5mut2 at concentrations as indicated for 72 hrs. The viable cells were quantified using MTT assay (A, B, C). (A) Effect of K5, K5mut1 and K5mut2 on the proliferation of primary HUVEC. (B) Effect of K5 on the proliferation of HepA cells, Bel7402 cells and Chang liver cells. (C) Effect of K5mut1 on the proliferation of HepA cells, Bel7402 cells and Chang liver cells. (D) Effect of K5mut2 on the proliferation of HepA cells, Bel7402 cells and Chang liver cells. (E) Quantitative analysis of HUVEC apoptosis induced by K5, K5mut1 and K5mut2. Apoptotic cell were quantified by flow cytometry with the negative control (PBS group) and the positive control (colchicines group). (F) Effect of K5, K5mut1 and K5mut2 on migration of primary HUVEC (top left: control; top right: K5; bottom left: K5mut1; bottom right: K5mut2). A modified Boyden Chamber-based assay was performed (8 μm pore sizes chamber was used). Values represent the mean of three determinations (±SD) as percentages of control (**: P < 0.05 versus control; ##: P < 0.05 K5 versus K5mut1).

Fig 4

K5mut1 exhibits more potent anti-hepatoma effect in grafted hepatocarcinoma mouse model. (A) Tumour tissues at day 14 treated with PBS, K5, K5mut1 and K5mut2, respectively. (B) An average of 62%, 75% and –7% suppression of primary tumour growth was observed in the K5-treated, K5mut1-treated and K5mut2-treated group compared with control group, respectively. Data are presented as mean ± SD. Values significantly lower than control are indicated (**: P < 0.05 versus control; ##: P < 0.05 K5 versus K5mut1). (C) Representative immunohistochemical data for CD31 immunoreactivity (×200 magnification). At the end of the study, a portion of each tumour from control and drug-treated (K5, K5mut1 and K5mut2) groups was processed for immunohistochemical staining for CD31 as described in ‘Materials and Methods’. a: PBS; b: K5; c: K5mut1; d: K5mut2. (D) Quantitative analysis. Data are the mean ± SD. Microvessels were counted from five randomly selected fields in tumours from three mice of each group (**: P < 0.05 versus control; ##: P < 0.05 K5 versus K5mut1).

Fig 5

K5mut1 exhibits more potent anti-hepatoma effect in hepatocarcinoma xenograft athymic mouse model. (A) Tumour tissues at day 30 treated with PBS, K5, K5mut1 and K5mut2, respectively. (B) An average of 68%, 77%, and 9% suppression of primary tumour growth was observed in the K5-treated, K5mut1-treated and K5mut2-treated group compared with control group, respectively. Data are presented as mean ± SD. Values significantly lower than control are indicated (**: P < 0.05 versus control; ##: P < 0.05 K5 versus K5mut1). (C) Representative immunohistochemical data for CD31 immunoreactivity (×200 magnification). A portion of each tumour from control and drug-treated (K5, K5mut1 and K5mut2) groups was processed for immunohistochemical staining for CD31 as described in ‘Materials and Methods’. a: PBS; b: K5; c: K5mut1; d: K5mut2. (D) Quantitative analysis. Data are the mean ± SD. Microvessels were counted from five randomly selected fields in tumours from three mice of each group (**: P < 0.05 versus control; ##: P < 0.05 K5 versus K5mut1).

Fig 6

K5mut3 with five serine residues replacing five acidic amino acids in NH2 terminal of K5 has enhanced activity as K5mut1. (A) Schematic diagram of K5mut3. Note that five of the acidic amino acids of K5 were substituted by Serine (arrows). (B) SDS-PAGE analysis of the purified K5mut3 protein with Coomassie blue staining. (C) Western blot analysis of the purified K5mut3 protein with antibody specific to His-tag. (D) Effects of K5mut3 on endothelial cell proliferation. Marker: Protein marker; Lane1: Total cell protein before IPTG induction; Lane2: Total cell protein before IPTG induction; Lane3: Purified recombinant protein of K5mut3.

Fig 7

CD spectra show that β-sheet is the structural feature for the active K5 and mutants. The far UV spectra show that K5 (▪)□K5mut1 (○) and K5mut3 (▿) have characteristic β-sheet spectrum. K5 and K5mut3 adopt the shape characteristic of a β-sheet with a single broad peak around 235 nm, while K5mut1 adopts the shape characteristic of a β-sheet with a single broad peak around 230 nm. The spectrum of K5mut2 (▵) is dominated by random coil structure.