Multiple, simultaneous, independent gradients for a versatile multidimensional liquid chromatography. Part II: Application 3 - Scouting optimization strategies for separation of monoclonal antibodies by dual simultaneous independent gradients of pH & salt on a weak cation exchange stationary phase.

In previous publications we have described the pISep dual simultaneous, independent gradients (DSIGs) liquid chromatography (LC) for uncoupling gradients of non-buffering solute (NaCl, urea or acetonitrile) from externally generated pH gradients. In DSIGs the shape and slope of the [salute] gradient does not depend on the shape and slope of the pH gradient. The technique allows in a single run true simultaneous two dimensional LC separation of complex protein mixtures on various stationary phases including anion, cation exchangers (AEX, CEX), reversed phase (RP), mixed mode and mixed bed. Using a humanized IgG1 (HIgG1) monoclonal antibody (MAb) and a variety of pH & [NaCl] DSIGs, we show that most of MAb isoforms can be successfully separated from each other. These experimental observations are supported by an initial theoretical argument presented here predicting an overall improvement of all MAb isoforms separation by DSIGs of pH & [NaCl]. Theoretical calculations predict that, in general, there exists an optimal non-zero isocratic salt concentration in a pH gradient separation that will resolve isoforms close in binding energy, but a wide range of salt concentrations will be required for acceptable resolution of all isoforms. Theory also predicts better separation of weaker rather than stronger binding isoforms. Experimentally, we have found that no one set of DSIGs LC conditions could optimally baseline resolve all identifiable MAb isoforms in a single run of reasonable duration. The versatility and simplicity of the pH & [NaCl] pISep DSIGs LC allows fast, automated scouting of protein separations over any range of pH from 2.4 to 10.8 and [NaCl] from 0 to 1 M without changing the chemistry of the buffering system. Due to the universal applicability of the pISep buffering system in IEX LC, the researcher is given a powerful tool to easily develop pH & [NaCl] DSIGs protocols that vary mobile phase compositions to achieve high resolution separations of targeted proteins.

Methanol gel electrophoresis: Separation of human fast and slow myosin light chain 1 and other myofibrillar protein isoforms on a single gel format.

This report describes a novel sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) resolving gel format that consistently yields the electrophoretic separation of the fast and slow isoforms of human sarcomeric myosin light chain 1 (MLC1). The inclusion of methanol as a constituent of the resolving gel impacted the electrophoretic mobility of proteins across a broad range of molecular masses. There was greater separation of the fast and slow isoforms of human MLC1, as well as separation and high resolution of fast and slow isoforms of the three myosin heavy chain isoforms that are expressed in human skeletal muscle on the same gel format. Furthermore, the same resolving gel format substantially altered the electrophoretic mobility of at least one isoform of tropomyosin in human striated muscle. It is possible that the inclusion of methanol in SDS-PAGE resolving gels could improve the separation of other proteins that are expressed in muscle and in other tissues and cell types.

The mRNA Expression and Regulation of Glucocorticoid Receptor Isoforms Associated with Response of Multiple Myeloma Treated with a Glucocorticoids-Dependent Regimen of Dexamethasone, Bortezomib and Thalidomide.

OBJECTIVE: Glucocorticoids (GCs) are the effective first-line drugs and indispensable in chemotherapy regimens to treat patients with multiple myeloma (MM). Previous studies in a variety of hematologic malignancies have shown that the biological action of GC is mediated through the expression and activation and of glucocorticoids receptor (GR) isoforms in vitro. GR and its regulation are crucial determinants of the efficacy of GC independent therapy. There is currently lack of research on patients with MM. METHODS: 132 patients with MM were divided into responders (78 cases) and nonresponders (54 cases) according to the efficacy evaluated after four cycles of GC-dependent regimen. 66 patients with iron-deficiency anemia were served as controls. Preparation of mononuclear bone marrow cells (MBMCs) was purified by Ficoll-Hypaque gradient centrifugation. The mRNA expression of GR α, ß, γ, P, SRp30, SRp40, HSP90, NF-κB and AP-1 were detected by real time RT-PCR. TRIAL REGISTRATION: CHiCTR-RCH-12002872. RESULTS: The expression of four GR isoforms exhibited the following trend in MM patients and controls: GRα>GR-P>GRγ>GRß. GRα and HSP90 expression in responders was significantly higher than that of the nonresponders (P<0.050). HSP90/GRα expression in MM patients exhibited significantly higher than that in controls (P<0.001). SRp30c and SRp40 mRNA expression both showed significant positive correlation with GRα transcript (P<0.001). Compared with controls, NF-kB and AP -1 expression in MM patients was higher. NF-kB and AP-1 expression of nonresponders were significantly higher than that of responders. The difference was not obvious statistically (P>0.050). CONCLUSION: Our findings raise the possibility that low expression of GRα and HSP90 plays important roles in nonresponders. Lack of HSP90 might affect GR structure and further take part in nonresponse. SRp30c and SRp40 mRNA expression both showed significant positive correlation with GRα. That might become new targets for treatment of nonresponders in MM patients, although further studies are needed for clarification.

Interfacial Activity of Lipoprotein (a) Isoforms with a Variable Number of Kringle IV Type 2 Repeats: A New Indicator of Cardiovascular Risk?

OBJECTIVE: Lipoprotein (a) [Lp(a)] is an LDL-like particle constituted by lipids, apolipoprotein B100 and apolipoprotein (a) [apo(a)], a multidomain glycoprotein whose molecular mass is dependent on the genetically encoded number of Kringle IV type 2 (KIV-2) repeats. Because Lp(a) isoforms have been associated with cardiovascular risk (CVR), we have investigated if their interfacial properties can contribute to distinguish between low and high-risk groups and thus be used as a new CVR indicator. METHODS: Four Lp(a) variants, each carrying a different apo(a) isoform (K20, K24, K25, and K29), were purified from plasma of homozygous donors and their interfacial properties characterized using ellipsometry and surface pressure techniques. RESULTS: Ellipsometry measurements revealed that these isoforms had a similar propensity to form adsorbed layers at hydrophobic-hydrophilic interfaces, but surface pressure enabled to clearly separate them into two groups: K20 and K24 on one side, and K25 and K29 on the other side. CONCLUSION: Though K24 and K25 differ only by a single KIV-2 domain, their sharp difference in surface pressure suggests a critical threshold between the two Lp(a) forms, providing insights into the use of condensed matter approaches to monitor CVR. Our findings may represent a new laboratory window to assist medical decisions and to develop precision medicine treatments, practices, and products for CVR, which can be extended to other cardiovascular disease conditions.

Synergism of in vitro plasmodicidal activity of phospholipase A2 isoforms isolated from panamanian Bothrops asper venom.

Bothrops asper is one of the most important snake species in Central America, mainly because of its medical importance in countries like Ecuador, Panama and Costa Rica, where this species causes a high number of snakebite accidents. Several basic phospholipases A2 (PLA2s) have been previously characterized from B. asper venom, but few studies have been carried out with its acidic isoforms. In addition, since snake venom is a rich source of bioactive substances, it is necessary to investigate the biotechnological potential of its components. In this context, this study aimed to carry out the biochemical characterization of PLA2 isoforms isolated from B. asper venom and to evaluate the antiparasitic potential of these toxins. The venom and key fractions were subjected to different chromatographic steps, obtaining nine PLA2s, four acidic ones (BaspAc-I, BaspAc-II, BaspAc-III and BaspAc-IV) and five basic ones (BaspB-I, BaspB-II, BaspB-III, BaspB-IV and BaspB-V). The isoelectric points of the acidic PLA2s were also determined, which presented values ranging between 4.5 and 5. The findings indicated the isolation of five unpublished isoforms, four Asp49-PLA, corresponding to the group of acidic isoforms, and one Lys49-PLA2-like. Acidic PLA2s catalyzed the degradation of all substrates evaluated; however, for the basic PLA2s, there was a preference for phosphatidylglycerol and phosphatidic acid. The antiparasitic potential of the toxins was evaluated, and the acidic PLA2s demonstrated action against the epimastigote forms of T. cruzi and promastigote forms of L. infantum, while the basic PLA2s BaspB-II and BaspB-IV showed activity against P. falciparum. The results indicated an increase of up to 10 times in antiplasmodial activity, when the Asp49-PLA2 and Lys49-PLA2 were associated with one another, denoting synergistic action between these PLA2 isoforms. These findings correspond to the first report of synergistic antiplasmodial action for svPLA2s, demonstrating that these molecules may be important targets in the search for new antiparasitic agents.

Identification and Clinical Associations of 3 Forms of Circulating T-cadherin in Human Serum.

CONTEXT: T-cadherin (T-cad) is a glycosylphosphatidylinositol (GPI)-anchored cadherin that mediates adiponectin to induce exosome biogenesis and secretion, protect cardiovascular tissues, promote muscle regeneration, and stimulate therapeutic heart protection by transplanted mesenchymal stem cells. CDH13, the gene locus of T-cad, affects plasma adiponectin levels most strongly, in addition to affecting cardiovascular disease risk and glucose homeostasis. Recently, it has been suggested that T-cad exists in human serum, although the details are still unclear. OBJECTIVE: To validate the existence of T-cad forms in human serum and investigate the association with clinical parameters of type 2 diabetes patients. METHODS: Using newly developed monoclonal antibodies against T-cad, pooled human serum was analyzed, and novel T-cad enzyme-linked immunosorbent assays (ELISAs) were developed. The serum T-cad concentrations of 183 Japanese type 2 diabetes patients were measured in a cross-sectional observational study. The main outcome measure was the existence of soluble T-cad in human serum. RESULTS: There were 3 forms of soluble T-cad: a 130-kDa form with a prodomain, a 100-kDa mature form, and a 30-kDa prodomain in human serum. Using newly developed ELISAs to measure them simultaneously, we found that the 130-kDa form of T-cad positively correlated with plasma adiponectin (r = 0.28, P < .001), although a physiological interaction with adiponectin was not observed in serum. The unique 30-kDa prodomain was associated with several clinical parameters in diabetes patients. CONCLUSION: We identified 3 novel forms of soluble T-cad. Their importance as disease markers and/or biomarkers of adiponectin function and the possible bioactivity of the respective molecules require further investigation.

Newly Identified Members of FGFR1 Splice Variants Engage in Cross-talk with AXL/AKT Axis in Salivary Adenoid Cystic Carcinoma.

Adenoid cystic carcinoma (ACC) is the second most common malignancy of the salivary gland. Although characterized as an indolent tumor, ACC often leads to incurable metastatic disease. Patients with ACC respond poorly to currently available therapeutic drugs and factors contributing to the limited response remain unknown. Determining the role of molecular alterations frequently occurring in ACC may clarify ACC tumorigenesis and advance the development of effective treatment strategies. Applying Splice Expression Variant Analysis and outlier statistics on RNA sequencing of primary ACC tumors and matched normal salivary gland tissues, we identified multiple alternative splicing events (ASE) of genes specific to ACC. In ACC cells and patient-derived xenografts, FGFR1 was a uniquely expressed ASE. Detailed PCR analysis identified three novel, truncated, intracellular domain-lacking FGFR1 variants (FGFR1v). Cloning and expression analysis suggest that the three FGFR1v are cell surface proteins, that expression of FGFR1v augmented pAKT activity, and that cells became more resistant to pharmacologic FGFR1 inhibitor. FGFR1v-induced AKT activation was associated with AXL function, and inhibition of AXL activity in FGFR1v knockdown cells led to enhanced cytotoxicity in ACC. Moreover, cell killing effect was increased by dual inhibition of AXL and FGFR1 in ACC cells. This study demonstrates that these previously undescribed FGFR1v cooperate with AXL and desensitize cells to FGFR1 inhibitor, which supports further investigation into combined FGFR1 and AXL inhibition as an effective ACC therapy.This study identifies several FGFR1 variants that function through the AXL/AKT signaling pathway independent of FGF/FGFR1, desensitizing cells to FGFR1 inhibitor suggestive of a potential resistance mechanism in ACC. SIGNIFICANCE: This study identifies several FGFR1 variants that function through the AXL/AKT signaling pathway independent of FGF/FGFR1, desensitizing cells to FGFR1 inhibitor, suggestive of a potential resistance mechanism in ACC.

Mass Spectrometric (MS) Analysis of Proteins and Peptides.

The human genome is sequenced and comprised of ~30,000 genes, making humans just a little bit more complicated than worms or flies. However, complexity of humans is given by proteins that these genes code for because one gene can produce many proteins mostly through alternative splicing and tissue-dependent expression of particular proteins. In addition, post-translational modifications (PTMs) in proteins greatly increase the number of gene products or protein isoforms. Furthermore, stable and transient interactions between proteins, protein isoforms/proteoforms and PTM-ed proteins (protein-protein interactions, PPI) add yet another level of complexity in humans and other organisms. In the past, all of these proteins were analyzed one at the time. Currently, they are analyzed by a less tedious method: mass spectrometry (MS) for two reasons: 1) because of the complexity of proteins, protein PTMs and PPIs and 2) because MS is the only method that can keep up with such a complex array of features. Here, we discuss the applications of mass spectrometry in protein analysis.

Multiplex LC-MS/MS for simultaneous determination of 25-hydroxyvitamin D, 24,25-dihydroxyvitamin D3, albumin, and vitamin D-binding protein with its isoforms: One-step estimation of bioavailable vitamin D and vitamin D metabolite ratio.

Bioavailable vitamin D and vitamin D metabolite ratio (VMR) have emerged as potential novel vitamin D markers. We developed a multiplex liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to determine all elements necessary for the calculation of bioavailable vitamin D and VMR, including 25-hydroxyvitamin D [25-(OH)D] and 24,25-dihydroxyvitamin D3 [24,25-(OH)2D3], VDBP and its isoforms, and albumin. Following separate reactions of hexane extraction and trypsin digestion, serum samples were analyzed using LC-MS/MS to measure 25-(OH)D3, 25-(OH)D2, 24,25-(OH)2D3, VDBP and its isoforms, and albumin. Analytical performances were assessed. Korean (n = 229), Arab (n = 98), White (n = 99) and Black American (n = 99) samples were analyzed. Bioavailable vitamin D and VMR were calculated. All target molecules were clearly separated and accurately quantified by LC-MS/MS. Analytical performances, including imprecision, accuracy, ion suppression, limit of quantification, linearity, and comparison with existing methods were within acceptable levels. The allele frequencies of VDBP isoforms in various races resulted similar to previously known values. The levels of bioavailable vitamin D were highest in White Americans and lowest in Black Americans. We have successfully developed a multiplex LC-MS/MS-based assay method that can simultaneously perform the measurement of all parameters needed to calculate bioavailable vitamin D and VMR. Our devised method was robust and reliable in terms of analytical performances and could be applied to routine clinical samples in the future to more accurately assess vitamin D status.

Regulatory mechanisms of tau protein fibrillation under the conditions of liquid-liquid phase separation.

One of the hallmarks of Alzheimer's disease and several other neurodegenerative disorders is the aggregation of tau protein into fibrillar structures. Building on recent reports that tau readily undergoes liquid-liquid phase separation (LLPS), here we explored the relationship between disease-related mutations, LLPS, and tau fibrillation. Our data demonstrate that, in contrast to previous suggestions, pathogenic mutations within the pseudorepeat region do not affect tau441's propensity to form liquid droplets. LLPS does, however, greatly accelerate formation of fibrillar aggregates, and this effect is especially dramatic for tau441 variants with disease-related mutations. Most important, this study also reveals a previously unrecognized mechanism by which LLPS can regulate the rate of fibrillation in mixtures containing tau isoforms with different aggregation propensities. This regulation results from unique properties of proteins under LLPS conditions, where total concentration of all tau variants in the condensed phase is constant. Therefore, the presence of increasing proportions of the slowly aggregating tau isoform gradually lowers the concentration of the isoform with high aggregation propensity, reducing the rate of its fibrillation. This regulatory mechanism may be of direct relevance to phenotypic variability of tauopathies, as the ratios of fast and slowly aggregating tau isoforms in brain varies substantially in different diseases.

Detecting protein and post-translational modifications in single cells with iDentification and qUantification sEparaTion (DUET).

While technologies for measuring transcriptomes in single cells have matured, methods for measuring proteins and their post-translational modification (PTM) states in single cells are still being actively developed. Unlike nucleic acids, proteins cannot be amplified, making detection of minute quantities from single cells difficult. Here, we develop a strategy to detect targeted protein and its PTM isoforms in single cells. We barcode the proteins from single cells by tagging them with oligonucleotides, pool barcoded cells together, run bulk gel electrophoresis to separate protein and its PTM isoform and quantify their abundances by sequencing the oligonucleotides associated with each protein species. We used this strategy, iDentification and qUantification sEparaTion (DUET), to measure histone protein H2B and its monoubiquitination isoform, H2Bub, in single yeast cells. Our results revealed the heterogeneities of H2B ubiquitination levels in single cells from different cell-cycle stages, which is obscured in ensemble measurements.

Engineering the Ovarian Hormones Inhibin A and Inhibin B to Enhance Synthesis and Activity.

Ovarian-derived inhibin A and inhibin B (heterodimers of common α- and differing ß-subunits) are secreted throughout the menstrual cycle in a discordant pattern, with smaller follicles producing inhibin B, whereas the dominant follicle and corpus luteum produce inhibin A. The classical function for endocrine inhibins is to block signalling by activins (homodimers of ß-subunits) in gonadotrope cells of the anterior pituitary and, thereby, inhibit the synthesis of FSH. Whether inhibin A and inhibin B have additional physiological functions is unknown, primarily because producing sufficient quantities of purified inhibins, in the absence of contaminating activins, for preclinical studies has proven extremely difficult. Here, we describe novel methodology to enhance inhibin A and inhibin B activity and to produce these ligands free of contaminating activins. Using computational modeling and targeted mutagenesis, we identified a point mutation in the activin ß A-subunit, A347H, which completely disrupted activin dimerization and activity. Importantly, this ß A-subunit mutation had minimal effect on inhibin A bioactivity. Mutation of the corresponding residue in the inhibin ß B-subunit, G329E, similarly disrupted activin B synthesis/activity without affecting inhibin B production. Subsequently, we enhanced inhibin A potency by modifying the binding site for its co-receptor, betaglycan. Introducing a point mutation into the α-subunit (S344I) increased inhibin A potency ~12-fold. This study has identified a means to eliminate activin A/B interference during inhibin A/B production, and has facilitated the generation of potent inhibin A and inhibin B agonists for physiological exploration.

A wealth of genotype-specific proteoforms fine-tunes hemoglobin scavenging by haptoglobin.

The serum haptoglobin protein (Hp) scavenges toxic hemoglobin (Hb) leaked into the bloodstream from erythrocytes. In humans, there are two frequently occurring allelic forms of Hp, resulting in three genotypes: Homozygous Hp 1-1 and Hp 2-2, and heterozygous Hp 2-1. The Hp genetic polymorphism has an intriguing effect on the quaternary structure of Hp. The simplest form, Hp 1-1, forms dimers consisting of two α1ß units, connected by disulfide bridges. Hp 2-1 forms mixtures of linear (α1)2(α2)n-2(ß)n oligomers (n > 1) while Hp 2-2 occurs in cyclic (α2)n(ß)n oligomers (n > 2). Different Hp genotypes bind Hb with different affinities, with Hp 2-2 being the weakest binder. This behavior has a significant influence on Hp's antioxidant capacity, with potentially distinctive personalized clinical consequences. Although Hp has been studied extensively in the past, the finest molecular details of the observed differences in interactions between Hp and Hb are not yet fully understood. Here, we determined the full proteoform profiles and proteoform assemblies of all three most common genetic Hp variants. We combined several state-of-the-art analytical methods, including various forms of chromatography, mass photometry, and different tiers of mass spectrometry, to reveal how the tens to hundreds distinct proteoforms and their assemblies influence Hp's capacity for Hb binding. We extend the current knowledge by showing that Hb binding does not just depend on the donor's genotype, but is also affected by variations in Hp oligomerization, glycosylation, and proteolytic processing of the Hp α-chain.

Purification and Assays of Tachylectin-2.

Tachylectin-2, a 27-kDa protein consisting of a five-bladed ß-propeller structure, is purified by three steps of chromatography, including dextran sulfate-Sepharose CL-6B, CM-Sepharose CL-6B, and Mono S. Three isolectins of tachylectin-2 including tachylectin-2a, -2b, and -2c are purified. These isolectins exhibit hemagglutinating activity against human A-type erythrocytes in a Ca2+-independent manner with tachylectin-2b showing the highest activity. Tachylectin-2b specifically agglutinates Staphylococcus saprophyticus KD. The tachylectin-2b-mediated hemagglutination is inhibited in the presence of GlcNAc and GalNAc. The association constants for GlcNAc and GalNAc are Ka = 1.95 × 104 M-1 and Ka = 1.11 × 103 M-1, respectively. Ultracentrifugation analysis shows that tachylectin-2b is present in monomer form in solution.

Molecular cloning and functional analysis of three STAT isoforms in red swamp crayfish Procambarus clarkii.

The Janus kinase (JAK)/signal transducers and activators of transcription (STAT) signaling pathway is associated with the innate immune system and plays crucial roles in the mediation of immune response to viral infections. In this study, three STAT isoform cDNAs were cloned from the red swamp crayfish Procambarus clarkii, and they were designated as PcSTATa, PcSTATb, and PcSTATc. PcSTATa and PcSTATb were generated through the alternative splicing of the last exon, and PcSTATc was produced by intron retention. PcSTATa, PcSTATb, and PcSTATc contained 2382, 2337, and 2274 bp open reading frames encoding proteins with 793, 778, and 757 amino acid residues, respectively. Domain prediction analysis revealed that three isoforms of PcSTATs contain a STAT interaction domain, a STAT all-alpha domain, a STAT DNA binding domain, and a Src-homology 2 domain. The mRNA transcripts of three PcSTAT isoforms were detected in all examined tissues of male and female crayfish. The expression levels of the three PcSTAT isoforms in the hemocytes, gills, and intestines significantly changed after the white spot syndrome virus (WSSV) challenge. PcSTAT silencing by dsRNA interference could positively regulate the expression levels of three anti-lipopolysaccharide factors (PcALF1, PcALF2, and PcALF6) and two crustins (PcCrus1 and PcCrus2) and negatively regulate the expression levels of three ALFs (PcALF3, PcALF4, and PcALF5) and two crustins (PcCrus3 and PcCrus4). These results suggest that all three PcSTAT isoforms are involved in the host defense against WSSV infection.

The Human Cytomegalovirus pUL145 Isoforms Act as Viral DDB1-Cullin-Associated Factors to Instruct Host Protein Degradation to Impede Innate Immunity.

Human cytomegalovirus (HCMV) causes diseases in individuals with immature or compromised immunity. To evade immune control, HCMV evolved numerous antagonists targeting the interferon system at multiple levels. By comparative analysis of naturally arising variants of the most widely studied HCMV strain, AD169, and a panel of targeted mutants, we uncover the UL145 gene as indispensable for STAT2 downregulation. Ribosome profiling confirms the translation of the canonical pUL145 protein (pUL145-Long) and newly identifies a shorter isoform (pUL145-Short). Both isoforms recruit DDB1-containing ubiquitin ligases to induce proteasomal degradation of STAT2. An alanine-scanning mutagenesis discloses the DDB1 interaction motif of pUL145 that resembles the DDB1-binding interface of cellular substrate receptors of DDB1-containing ubiquitin ligases. Thus, pUL145 constitutes a viral DDB1-cullin-associated factor (vDCAF), which mimics cellular DCAFs to exploit the ubiquitin-proteasome system to impede antiviral immunity. Notably, the viral exploitation of the cullins can be targeted to restore the efficacy of the host immune response.

Single Step Purification of Glycogen Synthase Kinase Isoforms from Small Scale Transient Expression in HEK293 Cells with a Calcium-Dependent Fragment Complementation System.

Purification of proteins for the biophysical analysis of protein interactions occurring in human cells can benefit from methods that facilitate the capture of small amounts of natively processed protein obtained using transient mammalian expression systems. We have used a novel calcium-dependent fragment complementation-based affinity method to effectively purify full length glycogen synthase kinase 3 (GSK3) α and ß isoforms to study their interaction with amyloid ß peptide (Aß42). Using these proteins, purified from 1 mg of total cell lysate, we measured an apparent KD of ≤100 pM between GSK3α/ß and immobilized Aß42 with surface plasmon resonance technology. This approach can be used to retrieve useful quantities of protein for biophysical experiments with small scale mammalian cell culture.

The TFF Peptides xP1 and xP4 Appear in Distinctive Forms in the Xenopus laevis Gastric Mucosa: Indications for Different Protective Functions.

The gastric secretory trefoil factor family (TFF) peptides xP1 and xP4 are the Xenopus laevis orthologs of mammalian TFF1 and TFF2, respectively. The aim of this study was to analyze the molecular forms of xP1 and xP4 in the X. laevis gastric mucosa by FPLC. xP1 mainly occurred in a monomeric low-molecular-mass form and only a minor subset is associated with the mucus fraction. The occurrence of monomeric xP1 is unexpected because of its odd number of cysteine residues. Probably a conserved acidic residue flanking Cys55 allows monomeric secretion. Furthermore, Cys55 is probably post-translationally modified. For the first time, we hypothesize that the free thiol of monomeric xP1-and probably also its mammalian ortholog TFF1-could have a protective scavenger function, e.g., for reactive oxygen/nitrogen species. In contrast, xP4 mainly occurs in a high-molecular-mass form and is non-covalently bound to a mucin similarly as TFF2. In vitro binding studies with radioactively labeled porcine TFF2 even showed binding to X. laevis gastric mucin. Thus, xP4 is expected to bind as a lectin to an evolutionary conserved sugar epitope of the X. laevis ortholog of mucin MUC6 creating a tight mucus barrier. Taken together, xP1 and xP4 appear to have different gastric protective functions.

Detection of black market follistatin 344.

Follistatin, a myostatin-inhibiting protein, is prohibited according to chapter S4 of the "WADA 2019 List of Prohibited Substances and Methods". While currently no approved pharmaceutical formulations of follistatin are available, follistatin can be bought on the black market. Most of the products are labeled "follistatin 344" (FS344), a few "follistatin 315". A study on FS344 black market products was performed and an electrophoretic detection method for serum and urine developed. While only nine of the 17 tested products actually contained follistatin, in some of the others growth promoting peptides were found (e.g. MGF, GHRP-2). Surprisingly, all nine products contained His-tagged FS344 and a high degree of its oligomers. The detection method is based on immunomagnetic purification followed by SDS-PAGE and Western blotting with a monoclonal anti-His antibody. Alternatively, a monoclonal anti-follistatin antibody can be used. For immunoprecipitation (IP), a polyclonal anti-follistatin antibody is applied. An evaluation of suitable antibodies for IP and immunoblotting is also presented. Furthermore, practically all currently available follistatin standards were investigated. The detection limit of the method for black market FS344 in urine is ca 0.1 ng/mL for 10 mL. For a sample volume of 100 µL, an LOD of 5 ng/mL could be achieved for serum. Due to the presence of His-tags an unambiguous differentiation from endogenous follistatin is possible.

Design and structural characterisation of olfactomedin-1 variants as tools for functional studies.

BACKGROUND: Olfactomedin-1 (Olfm1; also known as Noelin or Pancortin) is a highly-expressed secreted brain and retina protein and its four isoforms have different roles in nervous system development and function. Structural studies showed that the long Olfm1 isoform BMZ forms a disulfide-linked tetramer with a V-shaped architecture. The tips of the Olfm1 "V" each consist of two C-terminal ß-propeller domains that enclose a calcium binding site. Functional characterisation of Olfm1 may be aided by new biochemical tools derived from these core structural elements. RESULTS: Here we present the production, purification and structural analysis of three novel monomeric, dimeric and tetrameric forms of mammalian Olfm1 for functional studies. We characterise these constructs structurally by high-resolution X-ray crystallography and small-angle X-ray scattering. The crystal structure of the Olfm1 ß-propeller domain (to 1.25 Å) represents the highest-resolution structure of an olfactomedin family member to date, revealing features such as a hydrophilic tunnel containing water molecules running into the core of the domain where the calcium binding site resides. The shorter Olfactomedin-1 isoform BMY is a disulfide-linked tetramer with a shape similar to the corresponding region in the longer BMZ isoform. CONCLUSIONS: These recombinantly-expressed protein tools should assist future studies, for example of biophysical, electrophysiological or morphological nature, to help elucidate the functions of Olfm1 in the mature mammalian brain. The control over the oligomeric state of Olfm1 provides a firm basis to better understand the role of Olfm1 in the (trans-synaptic) tethering or avidity-mediated clustering of synaptic receptors such as post-synaptic AMPA receptors and pre-synaptic amyloid precursor protein. In addition, the variation in domain composition of these protein tools provides a means to dissect the Olfm1 regions important for receptor binding.