Background Biochemical bone turnover markers (BTM) are useful tools to assess bone remodeling at the cellular level. N-terminal propeptide of type I procollagen (PINP) has been recommended as a ...reference marker for bone formation in research studies. Methods We describe the results of a multicenter study for routine clinical laboratory assays for PINP in serum and plasma. Four centers (Athens, Greece GR, Copenhagen, Denmark DK, Liege, Belgium BE and Sheffield, United Kingdom UK) collected serum and plasma (EDTA) samples from 796 patients presenting to osteoporosis clinics. Specimens were analyzed in duplicate with each of the available routine clinical laboratory methods according to the manufacturers' instructions. Passing-Bablok regressions, Bland-Altman plots, V-shape evaluation method and the concordance correlation coefficient for PINP values between serum and plasma specimens and between methods were used to determine the agreement between results. A generalized linear model was employed to identify possible variables that affected the relationship between the methods. Results We showed that both EDTA plasma and serum were suitable for PINP determination. We observed a significant proportional bias between Orion radioimmunoassay and the automated methods for PINP (Roche Cobas and IDS iSYS), which both gave very similar results. The multivariate model did not improve the excellent correlation that was observed between the methods. Conclusions Harmonization of PINP assays is possible by applying a correction factor or correctly assigning the values of the calibrators. This work will benefit from further collaboration between assays manufacturers and clinical laboratory professionals.
•Propeptides are short sequences that facilitate protein folding.•Rhizomucor miehei lipase propeptide mutants exhibited increased activity.•Highly conserved sites in propeptide had a more significant ...impact on RML.•Our findings support a new idea for producing desired increases in activity.
The propeptide is a short sequence that facilitates protein folding. In this study, four highly active Rhizomucor miehei lipase (RML) mutants were obtained through saturation mutagenesis at three propeptide positions: Ser8, Pro35, and Pro47. The enzyme activities of mutants P35 N, P47 G, P47 N, and S8E/P35S/P47A observed at 40 °C, and pH 8.0 were 10.19, 7.53, 6.15, and 8.24 times of that wild-type RML, respectively. The S8E/P35S/P47A mutant showed good thermostability. After incubation at 40 °C for 1 h, 98.98 % of its initial activity remained, whereas wild-type RML retained only 78.76 %. This result indicated that the enhancement of hydrophilicity of 35- and 47- amino-acid residues could promote the interaction between the propeptide and the mature peptide and the enzyme activity and expression level. Highly conserved sites had a more significant impact on enzyme performance than did other sites, similar to the Pro35 and Pro47 mutants showed in this study. This study provides a new idea for protein modification: enzyme performance can be improved through propeptide regulation.
•Standardized preanalytical procedures are important to minimise variability of BTMs.•Performance of automated assays (PINP and CTX) are acceptable for routine practice.•As an alternative to BMD ...testing, BTMs may be useful to monitor osteoporosis therapies.•BTMs are not useful in diagnosis, or individual prediction of bone loss or fracture.•Harmonization of assays (PINP and CTX) would help improve interlaboratory agreement.
The adult bone is continuously being remodelled to repair microdamage, preserve bone strength and mechanical competence as well as maintain calcium homeostasis. Bone turnover markers are products of osteoblasts (bone formation markers) and osteoclasts (bone resorption markers) providing a dynamic assessment of remodelling (turnover). Resorption-specific bone turnover markers are typically degradation products of bone collagen molecules (N- NTX and C-telopeptide cross-linked type 1 collagen CTX), which are released into the circulation and excreted in urine; or enzymatic activities reflecting osteoclastic resorption, tartrate-resistant acid phosphatase TRACP. Formation-specific bone turnover markers embrace different osteoblastic activities: type 1 collagen synthesis (Procollagen type I N- propeptide PINP), osteoblast enzymes (bone-specific alkaline phosphatase BALP), or bone matrix proteins osteocalcin. Among individuals not receiving osteoporosis treatment, resorption and formation markers are tightly linked and highly correlated (r = 0.6–0.8).
Significant biological variability was reported in the past, but these issues have been greatly improved with automated assays and attention to pre-analytical and analytical factors that are known to influence bone turnover marker levels. Bone turnover markers are not useful in the diagnosis of osteoporosis, the individual prediction of bone loss, fracture, or rare complications, or in the selection of pharmacological treatment. Despite remaining issues with reference intervals and assays harmonization, bone turnover markers have proven to be useful in elucidating the pharmacodynamics and effectiveness of osteoporosis medications in clinical trials. As an alternative to BMD testing, BTMs may be useful to monitor osteoporosis therapies.
Key points
Fetal growth restriction increases the risk of fetal and neonatal mortality and morbidity, and contributes to increased risk of chronic disease later in life.
Intra‐amniotic insulin‐like ...growth factor‐1 (IGF1) treatment of the growth‐restricted ovine fetus improves fetal growth, but postnatal effects are unknown.
Here we report that intra‐amniotic IGF1 treatment of the growth‐restricted ovine fetus alters size at birth and mechanisms of early postnatal growth in a sex‐specific manner.
We also show that maternal plasma C‐type natriuretic peptide (CNP) products are related to fetal oxygenation and size at birth, and hence may be useful for non‐invasive monitoring of fetal growth restriction.
Intrauterine IGF1 treatment in late gestation is a potentially clinically relevant intervention that may ameliorate the postnatal complications of fetal growth restriction.
Placental insufficiency‐mediated fetal growth restriction (FGR) is associated with altered postnatal growth and metabolism, which are, in turn, associated with increased risk of adult disease. Intra‐amniotic insulin‐like growth factor‐1 (IGF1) treatment of ovine FGR increases growth rate in late gestation, but the effects on postnatal growth and metabolism are unknown. We investigated the effects of intra‐amniotic IGF1 administration to ovine fetuses with uteroplacental embolisation‐induced FGR on phenotypical and physiological characteristics in the 2 weeks after birth. We measured early postnatal growth velocity, amino‐terminal propeptide of C‐type natriuretic peptide (NTproCNP), body composition, tissue‐specific mRNA expression, and milk intake in singleton lambs treated weekly with 360 μg intra‐amniotic IGF1 (FGRI; n = 13 females, 19 males) or saline (FGRS; n = 18 females, 12 males) during gestation, and in controls (CON; n = 15 females, 22 males). There was a strong positive correlation between maternal NTproCNP and fetal oxygenation, and size at birth in FGR lambs. FGR lambs were ∼20% lighter at birth and demonstrated accelerated postnatal growth velocity. IGF1 treatment did not alter perinatal mortality, partially abrogated the reduction in newborn size in females, but not males, and reduced accelerated growth in both sexes. IGF1‐mediated upregulation of somatotrophic genes in males during the early postnatal period could suggest that treatment effects are associated with delayed axis maturation, whilst treatment outcomes in females may rely on the reprogramming of nutrient‐dependent mechanisms of growth. These data suggest that the growth‐restricted fetus is responsive to intra‐amniotic intervention with IGF1, and that sex‐specific somatotrophic effects persist in the early postnatal period.
Key points
Fetal growth restriction increases the risk of fetal and neonatal mortality and morbidity, and contributes to increased risk of chronic disease later in life.
Intra‐amniotic insulin‐like growth factor‐1 (IGF1) treatment of the growth‐restricted ovine fetus improves fetal growth, but postnatal effects are unknown.
Here we report that intra‐amniotic IGF1 treatment of the growth‐restricted ovine fetus alters size at birth and mechanisms of early postnatal growth in a sex‐specific manner.
We also show that maternal plasma C‐type natriuretic peptide (CNP) products are related to fetal oxygenation and size at birth, and hence may be useful for non‐invasive monitoring of fetal growth restriction.
Intrauterine IGF1 treatment in late gestation is a potentially clinically relevant intervention that may ameliorate the postnatal complications of fetal growth restriction.
Aim
Heart failure with preserved ejection fraction (HFpEF) is associated with myocardial remodelling including severe pro‐fibrotic changes contributing to an increase in left ventricular stiffness ...and diastolic dysfunction. Serum C‐terminal propeptide of procollagen type I (PIP) strongly correlates with the turnover of extracellular cardiac matrix proteins and fibrosis. Torasemide, but not furosemide, was described to reduce collagen type I synthesis in clinically unstable patients with heart failure with reduced ejection fraction. We evaluated whether its effect translated to HFpEF patients with type 2 diabetes mellitus (T2DM) and abnormal basal PIP levels.
Methods and results
We performed a relatively small, single‐centre, randomised, double‐blind, two‐arm parallel‐group, active controlled clinical trial in 35 HFpEF patients with T2DM to determine the effects of a 9‐month treatment with torasemide vs. furosemide on changes of serum PIP levels. Patients with increased PIP levels (≥110 ng/mL), or evidence of structural changes with a left atrial volume index (LAVI) >29 mL/m2 and abnormal PIP levels (≥70 ng/mL), were eligible to participate. Fifteen patients were female (42%), mean age was 69 years, body mass index was 34.7 kg/m2, 83% were in New York Heart Association class II/III. Echocardiographic characteristics showed a mean left ventricular ejection fraction of >60%, a left ventricular mass index >120 g/m2, an E/e' ratio of 14, and a LAVI of 40 mL/m2 with a NT‐proBNP of 174 ng/L and a 6‐minute walk distance of 421 m. Mean per cent change in PIP was 2.63 ± 5.68% (±SEM) in torasemide vs. 2.74 ± 6.49% in furosemide (P = 0.9898) treated patients. Torasemide was not superior to furosemide in improving functional capacity, diastolic function, quality of life, or neuroendocrine activation.
Conclusion
In this hypothesis‐generating, mechanistic trial in stable HFpEF patients with T2DM, neither long‐term administration of torasemide nor furosemide was associated with a significant effect on myocardial fibrosis, as assessed by serum PIP. Further studies are urgently needed in this field. More specific diuretic and anti‐fibrotic treatment strategies in T2DM and/or HFpEF are warranted.
Posttranslational modifications of proteins can impact their therapeutic efficacy, stability, and potential for pharmaceutical development. The Group AStreptococcus pyogenesC5a peptidase (ScpA) is a ...multi-domain protein composed of an N-terminal signal peptide, a catalytic domain (including propeptide), three fibronectin domains, and cell membrane-associated domains. It is one of several proteins produced by Group AS. pyogenesknown to cleave components of the human complement system. After signal peptide removal, ScpA undergoes autoproteolysis and cleaves its propeptide for full maturation. The exact location and mechanism of the propeptide cleavage, and the impact of this cleavage on stability and activity, are not clearly understood, and the exact primary sequence of the final enzyme is not known. A form of ScpA with no autoproteolysis fragments of propeptide present may be more desirable for pharmaceutical development from a regulatory and a biocompatibility in the body perspective. The current study describes an in-depth structural and functional characterization of propeptide truncated variants of ScpA expressed inEscherichia colicells. All three purified ScpA variants, ScpA, 79ΔPro, and 92ΔPro, starting with N32, D79, and A92 positions, respectively, showed similar activity against C5a, which suggests a propeptide-independent activity profile of ScpA. CE-SDS and MALDI top-down sequencing analyses highlight a time-dependent propeptide autoproteolysis of ScpA at 37 °C with a distinct end point at A92 and/or D93. In comparison, all three variants of ScpA exhibit similar stability, melting temperatures, and secondary structure orientation. In summary, this work not only highlights propeptide localization but also provides a strategy to recombinantly produce a final mature and active form of ScpA without any propeptide-related fragments.
Patients with schizophrenia with second-generation antipsychotics (SGAs) treatment have shown an increased risk of bone fragility and susceptibility to fracture; however, it is still unclear whether ...this risk is derived from the effect of antipsychotics on balance of bone metabolism.
We investigated the changes of two bone turnover biomarkers (BTMs) concentrations in people with schizophrenia receiving SGAs: procollagen type I aminoterminal propeptide (PINP) and C-terminal telopeptide of type I collagen (CTX-1) as BTMs of osteogenesis and bone resorption, respectively, to explore how antipsychotics contribute to bone fragility.
We recruited 59 Chinese male patients with schizophrenia (32 drug-naïve first-episode (DNFE) patients and 27 chronic patients) to undergo 8 weeks SGAs treatment. Fasting peripheral blood samples of pre- and posttreatment were collected, plasma levels of PINP and CTX-1 were measured.
The interaction effects of group and time on PINP and CTX-1 concentrations were found (P = .016 and P = .008). There was a significant decrease for both BTMs concentrations of the posttreatment compared to the pretreatment (P<.001 and P = .003). Chronic patients had significantly higher changes of BTMs concentrations compared to DNFE patients (P = .048 and P = .024). There was a positive correlation of the two BTMs of pretreatment with disease course in DNFE group (r = .37, P = .039;r = .38, P = .035) and a negative correlation of PINP of pretreatment with age in the chronic group (r=-.40, P = .039).
Long-term SGAs medication inhibited osteogenesis in a dose- and time-dependent manner and damaged the balance of bone formation and bone resorption.
Microbial transglutaminase (MTG) is a usable enzyme for biomacromolecule modification. In the present study, a “molecular chaperonin” strategy was developed to produce MTG in E. coli cytoplasm with ...high expression level and a “small molecule-mediated chemical modification” strategy was adopted to strip propeptide chaperonin efficiently during purification. Propeptide (Pro) was expressed separately as a chaperonin to facilitate MTG expression in E. coli cytoplasm with a yield up to 300 mg or about 9 kU from 1 L fed-batch culture. Furthermore, small molecular chemicals were applied to interfere the interaction between MTG and Pro. Chemical acetylation was identified as a suitable method to strip Pro resulting in pure MTG with high specific activity up to 49.6 U/mg. The purified acetylated MTG was characterized by MS analysis. The deconvoluted mass and Peptide Sequence Tags analysis confirmed acetylation on amino groups of MTG protein. Finally, the applications of obtained MTG were demonstrated via protein polymerization of bovine serum albumin and PEGylation of human interferon-α2b. Our method provides MTG with high purity and specific activity as well as unique merit with masked amino groups thus avoiding self-polymerization and cross-linking between MTG and substrates.
Many secretory proteins and peptides are synthesized as inactive precursors that in addition to signal peptide cleavage undergo post‐translational processing to become biologically active ...polypeptides. Precursors are usually cleaved at sites composed of single or paired basic amino acid residues by members of the subtilisin/kexin‐like proprotein convertase (PC) family. In mammals, seven members have been identified, with furin being the one first discovered and best characterized. Recently, the involvement of furin in diseases ranging from Alzheimer’s disease and cancer to anthrax and Ebola fever has created additional focus on proprotein processing. We have developed a method for prediction of cleavage sites for PCs based on artificial neural networks. Two different types of neural networks have been constructed: a furin‐specific network based on experimental results derived from the literature, and a general PC‐specific network trained on data from the Swiss‐Prot protein database. The method predicts cleavage sites in independent sequences with a sensitivity of 95% for the furin neural network and 62% for the general PC network. The ProP method is made publicly available at http://www.cbs.dtu.dk/services/ProP.
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