To investigate roles of short peptides in gastroschisis (GS), we comprehensively analyzed peptides in amniotic fluid (AF), creating a fetal lamb model of GS. We created GS in 4 fetal lambs at 60 days of gestation. Three GS and 4 normal fetuses were delivered at term (145 days) by cesarean section, when AF samples were collected. Short peptides in the AF samples were detected and identified by mass spectrometry. One of the identified peptides was synthesized and it’s functions were investigated. In total, 77 peptide peaks were detected in the AF samples. Of these, 12 peptides showed significantly different intensity between the GS and control groups. Three of the 12 peptides were identified. One of the identified peptides with high intensity in the GS group was amino acids (AA) 135-185 of lamb annexin 7 (ANX7). A synthesized peptide for AA168-211 of human ANX7, which corresponded to AA135-185 of lamb ANX7, decreased anti-inflammatory cytokine secretion from mesothelial cells by an cytokine array study. We report a unique AF peptide profile in a GS model. One of the peptides increased in GS was suggested to possess pro-inflammatory potential. These peptides would be related to the pathophysiology of GS.
Gastroschisis (GS) is a congenital disease characterized by an abdominal wall defect through which bowel protrudes. The defect is almost always located to the right of umbilicus [
There are several theories put forward to explain the bowel inflammation and the development of “peel” in GS, with exposure to AF being most commonly implicated [
Another important type of molecule would be short peptides. Short peptides are thought to be generated by physiological cleavage of precursor proteins and by pathological degradation of various proteins. The former includes essential bioactive peptides like substance P [
Lambs at 60 days of gestation were subjected to surgical operation to create experimental GS. The preoperative management and anesthetic techniques have been reported previously [14,15]. Under general anesthesia, the uterus was exposed through a left flank incision, and the fetuses (n=4) were exposed through a transverse hysterotomy. An incision was made in the left upper part of the fetal abdominal wall and the bowel was gently delivered through the incision. The fetuses were then returned to uterus, with the hysterotomy being closed in 2 layers with 3-0 Biosyn. The ewe’s abdominal wall was closed in layers with Biosyn or Polysorb (US Surgical, Tyco Healthcare, Tokyo, Japan). AF samples from normal lambs (n=4) were used as controls.
The fetuses were delivered at term (145 days) by cesarean section, when approximately 4 ml of AF samples were collected by a syringe and immediately cooled by dry ice and stored at -80°C.
This study was approval by the ethics committee of School of Medicine and Health Sciences, University of Otago, Wellington Animal Ethics Committee (Wellington, New Zealand Approval Number 2-12).
Peptides in the AF samples were purified using weak cation exchange (MB-WCX; Bruker Daltonics, Ettliongen, Germany) as described previously [
For the identification of peptides of interest, MS/MS data of the peptides obtained by ESI-ion trap mass spectrometry was subjected to the searching program of MASCOT MS/MS ion search (Matrix Science, London, UK) against the Sheep Gene Index. Peptide identification was accepted when MASCOT search results delivered significant molecular weight search (MOWSE) scores (p<0.05).
For detection of lamb annexin A7 (ANX7), rabbit anti-human ANX7 polyclonal antibodies (Lifespan Biosciences, WA, USA) were used, since no antibody to lamb ANX7 was available. In a preliminary experiment, we confirmed that the antibody reacted to lamb ANX7 (data not shown). Proteins extracted from AF samples were separated by 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and then were transferred onto a poly vinylidene fluoride (PVDF) membrane. After blocking for 1 hour in a blocking buffer (TBS containing 2% ECL prime blocking agent (GE healthcare) and 0.1% Tween-20), the membrane was reacted to the anti-ANX7 antibodies at a dilution of 1:500 for 1 hour. After washing, bound antibodies were reacted with horseradish peroxidase (HRP)-conjugated goat antirabbit IgG antibodies (Dako, Glostrup, Denmark) and the substrate of HRP using ImmunoStar LD (Wako Co., Osaka, Japan). Finally, immunoreactive bands were detected using an image analyzer (LAS3000 Versatile Imaging System Fujifilm, Tokyo, Japan).
A peptide corresponding to AA168-211 of human annexin A7 was synthesized and named as hA7 (168-211) The region of hA7 (168-211) is a homologous part of AA135-185 of lamb annexin7.
Human adult mesothelial (HAM) cells were purchased from Zenbio (CA, USA). HAM cells, seeded on type1 collagen-coated dishes with a diameter of 10 cm, were cultured at 37° in normoxic air with 5% CO2 in Mesothelial Cell Growth Medium (ZEN-bio, CA, USA). After HAM cells were cultured in a starvation medium (Medium 199, Life Technologies, CA, US), 2% Penicillin G(10000 unit/ml)/ Storeptomycin(10 mg/ml)(SIGMA, UK), 10% charcoal dextran-treated Fetal bovine serum (Thermo SCIENTIFIC, MA, US)) for 24 hours, the cells were further incubated with or without 400n mol/ml of hA7(168211) for 48 hours. Finally, supernatant of the cell culture was collected and subjected to cytokine array analysis using a commercially available kit of Human Cytokine Array Panel A (R&D systems, Inc, MN, USA), which contained 36 different cytokines (complement component C5a, CD40 ligand, G-CSF, CXCL1/GRO α, CCL1/I-309, sICAM-1, IFN-γ, IL-1 α, IL-1 β, IL-1Ra, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12 p70, IL-13, IL-16, IL-17, IL-17E, IL-23, IL-27, IL-32 α, CXCL10/IP-10, CXCL11/I-TAC, CCL2/MCP-1, MIF, CCL3/MIP-1 alpha, CCL4/MIP-1 β, CCL5/RANTES, CXCL12/SDF-1, Serpin E1/PAI-1, TNF-α, TREM-1).
Differences of the peptide ion intensity between the GS and control groups were calculated by Student’s t-test. P<0.05 was considered to be significant.
We created experimental GS using 4 lamb fetuses at 60 days of gestation, as shown in
We then purified peptides from each of the AF samples using MB-WCX and determined peptide profiles of the AF samples in the range of 2000-10000 m/z by MALDI-TOF-MS. In this experiment the AF sample from the GS number 4 lamb that did not posses GS was omitted. A total of 77 peptide peaks were detected in the AF samples. The peptide profiles of the individual lamb fetuses and the average peptide profiles of the GS (n=3) and control (n=4) groups are shown in
We then compared the intensity of the 77 peptide peaks detected between the GS group and the controls. The intensity of 12 peptide peaks showed significant differences between the two groups (
Group | Number | Body weight |
Crown-to-rump length |
GS | AF protein concentration |
---|---|---|---|---|---|
GS | 1 | 3860 | 45 | + | 5.32 |
2 | 1890 | 37 | + | 4.84 | |
3 | 3300 | 45 | + | 1.00 | |
4 | 3472 | 44 | - | 4.40 | |
mean±SD | 3130.5 ± 859.5 | 42.8 ± 3.86 | 3.89 ± 1.96* | ||
Control | 1 | 1966 | 34 | - | 0.39 |
2 | 1966 | 44 | - | 0.87 | |
3 | 2766 | 44 | - | 0.25 | |
4 | 2976 | 37 | - | 0.51 | |
mean±SD | 2418.5 ± 529.5 | 39.8 ± 5.06 | 0.54 ± 0.31 |
GS=gastroschisis, AF=amniotic fluid, SD=standard deviation, *=p<0.05 (t-test).
We then tried to identify the 12 peptides by mass spectrometry and protein data base searching. As a result, we were able to identify 3 out of the 12 peptides, which were amino acid residues (AA) 135-185 of ANX7, AA163-218 of nuclear pore glycoprotein p62 (NUP62), and AA89-153 of ubiquitin fusion degradation protein 1 homolog (UFD1), as shown in
Difference (x) | Numbers of peptide peaks | |
---|---|---|
(fold change, GS/control) | average alone | with statistical significance |
3.0<x | 15 | 6 |
2.0<x | 23 | 9 |
1.5<x | 31 | 9 |
1.5≤x≤1/1.5 | 24 | 0 |
x<1/1.5 | 22 | 3 |
x<1/2.0 | 13 | 2 |
x<1/3.0 | 6 | 1 |
total peptide peaks | 77 | 12 |
Peptide |
Difference* | Identified amino acid sequences |
Theoretical |
Original protein | Mascot |
---|---|---|---|---|---|
5486.6 | 5.54 | GQPPYPSQPAAMTQGTQGTIRPAANFDAMRDAEVLRKAMKGFGTDEQAIIDV (135aa-185aa) | 5487.8 | annexin A7 |
75 |
5447.1 | 4.99 | TTTTTTTTTTTSTSTTGFSLNIKPLTPAGIPSNTAASGSAPSGPSVAAGGSGSAALT (163aa-218aa) | 5449.5 | nuclear pore glycoprotein p62 |
71 |
6867.3 | 2.22 | SGEPRASGARLRRLPLRGGVGGPHRQRSPAQRSGRARRRRPPCSPSTCSTTRSPGSSRAASPRSTA (89aa-153aa) | 6870.6 | ubiquitin fusion degradation protein 1 homolog |
65 |
m/z= mass/electric charge ratio, aa=amino acid, *=Fold change of GS/Control in intensities
As above, we demonstrated the increased intensity of 12 peptides in AF of fetal lambs with GS and identified 3 out of them. Focusing on one of the 3 peptides, which was assigned to AA135-185 of lamb ANX7, we extended our study. First, we investigated whether the amount of intact ANX7 molecules was increased or not by western blotting. As a result, the intact ANX7 with its expected molecular weight (MW) of 49.9 kDa was detected both in the GS and normal AF samples (
We then examined whether the region of AA135-185 lamb of ANX7 affect functions of cells. For this aim, we synthesized a peptide for AA168-211 of human ANX7 (hA7(168-211)), which corresponded to the region of AA135-185 of lamb ANX7 and examined effects of the synthesized peptide on secretion of various cytokines and soluble factors from HAM cells.
We cultured HAM cells with or without hA7 (168-211) and then the culture supernatant was applied to a cytokine array. As a result, 10 soluble factors (sICAM-1, IL-Ra, CXCL-1, IL-6, IL-8, IL-13, IL27, MCP-1, MIF, and serpin E1) were positively detected in hA7 (168-211)-stimulated or non-stimulated panels. (
In this study, to clarify the pathophysiology of the “peel” found in some infants with GS, we created a fetal lamb model of GS and comprehensively analyzed peptides in AF. Until now, no report has been available on peptide profiles of AF of GS both in humans and experimental animal models. Our findings are as follows: first, 3 of the 4 GS in which GS creation was attempted, displayed GS at term. Second, the intensity of 12 out of the detected 77 peptides in AF showed significant difference between the GS and control groups. Third, three out of the 12 peptides were identified as AA135-185 of ANX7, AA163218 of NUP62, and AA89-153 of UFD1. Fourth, the levels of the intact ANX7 showed no significant difference between the GS and control groups.
On the first point, we tried to create a GS model using 4 lamb fetuses, not all of the operated fetuses showed GS at term. This suggests that a simple incision of the abdominal wall in an early-gestation lamb fetus can heal over, unless sufficient bowel is delivered through the defect. The abdominal wall defect observed in patients with GS would involve an additional factor which maintains the abdominal wall defect and GS. Alternatively, the time to create experimental GS may be critical. Here, the experimental GS was created at 60 days of gestation as our first trial, since we successfully created an obstructive uropathy lamb model at the same days of gestation previously [
On the second point, the intensity of 12 out of the detected 77 peptides in AF was different between the GS and control groups. This indicates that profiles of proteins contained in AF, which are the source of degraded peptides, are skewed in GS. Alternatively, profiles of proteases that actually produce degraded peptides could be altered. Considering the points that amounts of proteins in AF has been reported to be increased in patients with GS [
On the third and fourth points, we successfully identified 3 out of the 12 peptides. This identification ratio in this study was lower than is usual in our human studies. This is probably due to the fact that the size of the protein data bases in lambs was much smaller than in that in humans, which would hamper effective protein identification.
One of the parent proteins of the identified three peptides was ANX7. In humans, production of ANX7 was demonstrated in liver, kidney, spleen, and placenta [
In our study, the increase of AA135-185 of ANX7 was demonstrated. In the production of this peptide derived from ANX7, certain endopeptidases that cleave ANX7 between Gly135 and Val185 should be involved. We therefore searched for such endopeptidases using a program MEROPS (http://merops.sanger.ac.uk/). However, we found no endopeptidases for production of the peptide by cleavage of ANX7 (data not shown). The mechanism for the production AA 135185 needs to be clarified in the future.
Another interest is whether the peptide of AA135-185 of ANX7 possesses bioactivity. We previously demonstrated that AC13 which corresponded to the C-terminal 13AA of apolipoprotein A enhanced IL-6 and IL-8 secretion from endothelial cells [
Parent proteins of the other two identified peptides were NUP62 and UFD1. NUP62 is an essential component of the nuclear pore complex [
In conclusion, we have created a fetal lamb model of GS in this study. We detected 12 peptides that were increased in the AF samples the of GS model, compared to the control and identified 3peptides. A human homolog of one of the increased AF peptides, hA7 (168-211), was found to have pro-inflammatory potential. The peptides increased in the GS model would be involved in the pathophysiology of the “peel” seen in GS.
This study was supported by the Japanese Society for Grant aid for Scientific Research (C). Suture materials were supplied by Covidience, Tokyo, Japan. These experiments would not have been possible without the skill and dedication of Doug Jensen. He consistently produces animals of timed gestation with the time of mating recorded to within 24 hours. The authors thank Ms Michiyo Yokoyama, Ms Junko Asano and Ms Atsuko Nozawa for their technical assistance.