Identification of Placental Transforming Growth Factor-β and Bikunin Metabolites as Contaminants of Pharmaceutical Human Chorionic Gonadotrophin Preparations by Proteomic Techniques

Malatos, S; Neubert, Hendrik; Kicman, Andrew T.; Iles, Ray K.

doi:10.1074/mcp.m500085-mcp200

Cited by 7 publications

(4 citation statements)

References 44 publications

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“…Furthermore, these glycoform differences are reflected in hCG’s major urinary metabolite hCGβcf, which can be detected by MALDI-ToF mass spectrometry [ 22 , 25 ]. However, it was presumed that purification and often concentration of the urine sample would be necessary [ 35 , 36 ]. Contrary to this, since hCGβcf is the most abundant glycoprotein in maternal urine we found that neat and preferably dilution of maternal urine resulted in reproducible mass spectral profiles in the mass regions expected for hCGβcf.…”

Section: Resultsmentioning

confidence: 99%

Direct and rapid mass spectral fingerprinting of maternal urine for the detection of Down syndrome pregnancy

et al. 2015

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BackgroundThe established methods of antenatal screening for Down syndrome are based on immunoassay for a panel of maternal serum biomarkers together with ultrasound measures. Recently, genetic analysis of maternal plasma cell free (cf) DNA has begun to be used but has a number of limitations including excessive turn-around time and cost. We aimed to develop an alternative method based on urinalysis that is simple, affordable and accurate.Method101 maternal urine samples sampled at 12–17 weeks gestation were taken from an archival collection of 2567 spot urines collected from women attending a prenatal screening clinic. 18 pregnancies in this set subsequently proved to be Down pregnancies. Samples were either neat urine or diluted between 10 to 1000 fold in dH2O and subjected to matrix assisted laser desorption ionization (MALDI), time of flight (ToF) mass spectrometry (MS). Data profiles were examined in the region 6,000 to 14,000 m/z. Spectral data was normalised and quantitative characteristics of the profile were compared between Down and controls.ResultsIn Down cases there were additional spectral profile peaks at 11,000-12,000 m/z and a corresponding reduction in intensity at 6,000-8,000 m/z. The ratio of the normalised values at these two ranges completely separated the 8 Down syndrome from the 39 controls at 12–14 weeks. Discrimination was poorer at 15–17 weeks where 3 of the 10 Down syndrome cases had values within the normal range.ConclusionsDirect MALDI ToF mass spectral profiling of maternal urinary has the potential for an affordable, simple, accurate and rapid alternative to current Down syndrome screening protocols.

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Section: Resultsmentioning

confidence: 99%

Direct and rapid mass spectral fingerprinting of maternal urine for the detection of Down syndrome pregnancy

et al. 2015

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“…These factors were initially termed antiviral lysozyme-C or antiviral RNases [70,71,72,73,74]. The appreciation that bikunin possessed anti-viral activity became apparent when a 15.8 kDa fragment of bikunin was identified as a contaminant in hCG preparations which inhibited the spread of KS lesions in HIV infections [75].…”

Section: Discussionmentioning

confidence: 99%

A Retrospective Analysis of the Cartilage Kunitz Protease Inhibitory Proteins Identifies These as Members of the Inter-α-Trypsin Inhibitor Superfamily with Potential Roles in the Protection of the Articulatory Surface

Smith

Melrose

2019

IJMS

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Aim: The aim of this study was to assess if the ovine articular cartilage serine proteinase inhibitors (SPIs) were related to the Kunitz inter-α-trypsin inhibitor (ITI) family. Methods: Ovine articular cartilage was finely diced and extracted in 6 M urea and SPIs isolated by sequential anion exchange, HA affinity and Sephadex G100 gel permeation chromatography. Selected samples were also subjected to chymotrypsin and concanavalin-A affinity chromatography. Eluant fractions from these isolation steps were monitored for protein and trypsin inhibitory activity. Inhibitory fractions were assessed by affinity blotting using biotinylated trypsin to detect SPIs and by Western blotting using antibodies to α1-microglobulin, bikunin, TSG-6 and 2-B-6 (+) CS epitope generated by chondroitinase-ABC digestion. Results: 2-B-6 (+) positive 250, 220,120, 58 and 36 kDa SPIs were detected. The 58 kDa SPI contained α1-microglobulin, bikunin and chondroitin-4-sulfate stub epitope consistent with an identity of α1-microglobulin-bikunin (AMBP) precursor and was also isolated by concanavalin-A lectin affinity chromatography indicating it had N-glycosylation. Kunitz protease inhibitor (KPI) species of 36, 26, 12 and 6 kDa were autolytically generated by prolonged storage of the 120 and 58 kDa SPIs; chymotrypsin affinity chromatography generated the 6 kDa SPI. KPI domain 1 and 2 SPIs were separated by concanavalin lectin affinity chromatography, domain 1 displayed affinity for this lectin indicating it had N-glycosylation. KPI 1 and 2 displayed potent inhibitory activity against trypsin, chymotrypsin, kallikrein, leucocyte elastase and cathepsin G. Localisation of versican, lubricin and hyaluronan (HA) in the surface regions of articular cartilage represented probable binding sites for the ITI serine proteinase inhibitors (SPIs) which may preserve articulatory properties and joint function. Discussion/Conclusions: The Kunitz SPI proteins synthesised by articular chondrocytes are members of the ITI superfamily. By analogy with other tissues in which these proteins occur we deduce that the cartilage Kunitz SPIs may be multifunctional proteins. Binding of the cartilage Kunitz SPIs to HA may protect this polymer from depolymerisation by free radical damage and may also protect other components in the cartilage surface from proteolytic degradation preserving joint function.

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“…Besides AC, ITI is also expressed in brain, placenta, liver, heart, lung, kidney and IVD and may be of importance in organ development (19,38). in HIV infections (83).…”

Section: Iti/bikunin Is a Multifunctional Proteinmentioning

confidence: 99%

A Retrospective Analysis of the Cartilage and Intervertebral Disc Kunitz Protease Inhibitory Proteins Identifies These as Members of The Inter-α-Trypsin Inhibitor Superfamily with potential roles in the protection of the articulatory surface

Smith¹,

Melrose²

2018

Preprint

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The aim of this study was to assess if the ovine articular cartilage serine proteinase inhibitors (SPIs) were related to the Kunitz inter-α-trypsin inhibitor (ITI) family. Ovine articular cartilage was finely diced and extracted in 6M urea and SPIs isolated by sequential anion exchange, HA affinity and Sephadex G100 gel permeation chromatography. Selected samples were also subjected to chymotrypsin and concanavalin-A affinity chromatography. Eluant fractions from these isolation steps were monitored for protein and trypsin inhibitory activity and pooled fractions assessed by affinity blotting using biotinylated trypsin to detect active SPIs and by Western blotting using antibodies to α1-microglobulin, bikunin, TSG-6 and 2-B-6 (+) CS stub epitope generated by chondroitinase-ABC digestion. This identified 2-B-6 (+) positive 220-250,120, 58 and 36 kDa SPIs. The 58 kDa SPI contained α1-microglobulin, bikunin and chondroitin-4-sulphate stub epitope consistent with its identity as the α1-microglobulin-bikunin (AMBP) precursor and was also isolated by concanavalin-A lectin affinity chromatography indicating it had N-glycosylation. Kunitz protease inhibitor (KPI) species of 36, 26, 12 and 6 kDa could be autolytically generated by prolonged storage of the aforementioned 120 and 58 kDa SPIs; chymotrypsin affinity chromatography also generated the 6kDa SPI. KPI domain 1 and 2 SPIs were separated by concanavalin lectin affinity chromatography, domain 1 displayed affinity for this lectin indicating it had N-glycosylation. KPI 1 and 2 both displayed potent inhibitory activity towards trypsin, chymotrypsin, kallikrein, leucocyte elastase and cathepsin G. Localisation of versican, lubricin and HA in the surface regions of articular cartilage represented probable binding sites for the ITI SPs with likely importance in the preservation of joint function.

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Identification of Placental Transforming Growth Factor-β and Bikunin Metabolites as Contaminants of Pharmaceutical Human Chorionic Gonadotrophin Preparations by Proteomic Techniques

Cited by 7 publications

References 44 publications

Direct and rapid mass spectral fingerprinting of maternal urine for the detection of Down syndrome pregnancy

Direct and rapid mass spectral fingerprinting of maternal urine for the detection of Down syndrome pregnancy

A Retrospective Analysis of the Cartilage Kunitz Protease Inhibitory Proteins Identifies These as Members of the Inter-α-Trypsin Inhibitor Superfamily with Potential Roles in the Protection of the Articulatory Surface

A Retrospective Analysis of the Cartilage and Intervertebral Disc Kunitz Protease Inhibitory Proteins Identifies These as Members of The Inter-α-Trypsin Inhibitor Superfamily with potential roles in the protection of the articulatory surface

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