Alpha-1-microglobulin/bikunin Precursor Research Articles

Urine biomarkers can predict prostate cancer and PI-RADS score prior to biopsy

Prostate-Specific Antigen (PSA) based screening of prostate cancer (PCa) needs refinement. The aim of this study was the identification of urinary biomarkers to predict the Prostate Imaging—Reporting and Data System (PI-RADS) score and the presence of PCa prior to prostate biopsy. Urine samples from patients with elevated PSA were collected prior to prostate biopsy (cohort = 99). The re-analysis of mass spectrometry data from 45 samples was performed to identify urinary biomarkers to predict the PI-RADS score and the presence of PCa. The most promising candidates, i.e. SPARC-like protein 1 (SPARCL1), Lymphatic vessel endothelial hyaluronan receptor 1 (LYVE1), Alpha-1-microglobulin/bikunin precursor (AMBP), keratin 13 (KRT13), cluster of differentiation 99 (CD99) and hornerin (HRNR), were quantified by ELISA and validated in an independent cohort of 54 samples. Various biomarker combinations showed the ability to predict the PI-RADS score (AUC = 0.79). In combination with the PI-RADS score, the biomarkers improve the detection of prostate carcinoma-free men (AUC = 0.89) and of those with clinically significant PCa (AUC = 0.93). We have uncovered the potential of urinary biomarkers for a test that allows a more stringent prioritization of mpMRI use and improves the decision criteria for prostate biopsy, minimizing patient burden by decreasing the number of unnecessary prostate biopsies.

ITRAQ-based mass spectrometry screen to identify serum biomarkers in systemic lupus erythematosus

ObjectiveSystemic lupus erythematosus (SLE) is a complex systemic autoimmune disorder with no reliable serum biomarkers currently available other than autoantibodies.MethodsIn the present study, isobaric tags for relative and absolute quantitation-based...

Plasma tumour and metabolism related biomarkers AMBP, LPL and Glyoxalase I differentiate heart failure with preserved ejection fraction with pulmonary hypertension from pulmonary arterial hypertension

BackgroundDiscrimination of heart failure with preserved ejection fraction with pulmonary hypertension (HFpEF-PH) from pulmonary arterial hypertension (PAH) is crucial for clinical management but may be challenging due to similarities in clinical and comorbid characteristics. We aimed to investigate tumour and metabolism related proteins in differentiating HFpEF-PH from PAH. MethodsSixty-nine tumour and metabolism plasma proteins were analysed with proximity extension assay in heathy controls (n = 20), patients with PAH (n = 48) and LHF-PH (n = 67) [HFpEF-PH (n = 31) and HF reduced EF-PH (n = 36)]. Haemodynamics were assessed with right heart catheterization. ResultsThe plasma levels of alpha-1-microglobulin/bikunin precursor (AMBP) and lipoprotein lipase (LPL), were higher in HFpEF-PH compared to healthy controls (p < 0.01), HFrEF-PH (p < 0.05), and PAH (p < 0.001). Glyoxalase I levels were higher in HFpEF-PH and HFrEF-PH compared to controls (p < 0.001) and PAH (p < 0.001). Each of plasma AMBP, LPL, and glyoxalase I, adjusted for age and sex in multivariable logistic regression models, could differentiate HFpEF-PH from PAH, with areas under the receiver operating characteristic curve (AUC) of 0.81, 0.84 and 0.79, respectively. The combination of AMBP, LPL and glyoxalse I yielded the largest AUC of 0.87 [95% confidence interval (0.79–0.95)] in discriminating HFpEF-PH from PAH, with a sensitivity of 87.1% and a specificity of 85.4%. In HFpEF-PH, the plasma levels of AMBP correlated with pulmonary arterial wedge pressure (rs = −0.42, p = 0.018). ConclusionsPlasma AMBP, LPL and glyoxalase I may facilitate the distinction of HFpEF-PH from PAH. Larger clinical studies are encouraged to confirm and validate our findings.

Identification of Novel Proteins Interacting with Proprotein Convertase Subtilisin/Kexin 9.

High levels of cholesterol, especially as low-density lipoprotein (LDL), are a well-known risk factor for atherosclerotic-related diseases. The key atherogenic property of LDL is its ability to form atherosclerotic plaque. Proprotein convertase subtilisin/kexin-9 (PCSK9) is an indirect regulator of plasma LDL levels by controlling the number of LDL receptor molecules expressed at the plasma membrane, especially in the liver. Herein, we performed a combination of affinity chromatography, mass spectrometry analysis and identification, and gene expression studies to identify proteins that interact with PCSK9. Through these studies, we identified three proteins, alpha-1-antitrypsin (A1AT), alpha-1-microglobulin/bikunin precursor (AMBP), and apolipoprotein H (APOH) expressed by C3A cells that interact with PCSK9. The expression levels of A1AT and APOH increased in cells treated with MITO+ medium, a condition previously shown to affect the function of PCSK9, as compared to treating with Regular (control) medium. However, AMBP expression did not change in response to the treatments. Additional studies are required to determine which of these proteins can modulate the expression/function of PCSK9. The identification of endogenous modulators of PCSK9’s function could lead to the development of novel diagnostic tests or treatment options for patients suffering hypercholesterolemia in combination with other chronic metabolic diseases.

Rapid, proteomic urine assay for monitoring progressive organ disease in Fabry disease

BackgroundFabry disease is a progressive multisystemic disease, which affects the kidney and cardiovascular systems. Various treatments exist but decisions on how and when to treat are contentious. The current marker...

The Effect of AMBP SNPs, Their Haplotypes, and Gene-Environment Interactions on the Risk of Atherothrombotic Stroke Among the Chinese Population.

Background/Objectives: Ischemic stroke (IS) is a severe and complex disorder with high morbidity and mortality rates and it has been associated with both environmental and genetic predisposing factors. The purpose of this study was to evaluate the association of the alpha-1-microglobulin/bikunin precursor (AMBP) gene polymorphisms with IS and any possible interactions between specific AMBP alleles and traditional risk factors among a Han Chinese cohort. Materials and Methods: We conducted a candidate gene study designed to characterize nine (9) single nucleotide polymorphisms (SNPs) of the AMBP gene among 195 patients with atherothrombotic stroke (ATS) (a major subtype of IS) and 184 nonstroke controls. Allelic and genotypic frequency differences were evaluated using a logistic regression model. False discovery rate (FDR) correction for multiple comparisons was used. The interactional analyses were performed using the multifactor dimensionality reduction test. Results: We found an association between the rs2567698 CC genotype (odds ratio [OR], 95% confidence interval [CI]: 2.176, 1.159-4.086) and the T allele (OR, 95% CI: 0.654, 0.446-0.960) with risk of ATS in men. However, these associations did not survive FDR correction. In haplotype analyses, the GCCCCCCCC haplotype had a higher frequency (OR, 95% CI: 2.191, 1.048-4.580) in ATS in the ≥45 years of age subgroup, whereas the GCCTCCCCC haplotype decreased the risk for ATS (OR, 95% CI: 0.543, 0.345-0.853) in men. In addition, we also found interactions for ATS risk between SNPs in the AMBP gene and modifiable risk factors for ATS, including: rs11788411 and hypertension in the overall population and women; rs2251680 and hypertension in subjects aged 45 years and older, as well as the interaction among hypertension and the rs2567698 and rs10817564 genotypes in men. Conclusion: Our results show a possible association between AMBP SNP haplotypes and gene-environment interactions with ATS susceptibility in a Han Chinese cohort.

Quantitative Urinary Proteome Reveals Potential Biomarkers for Ureteropelvic Junction Obstruction.

Ureteropelvic junction obstruction (UPJO) is a common obstructive disease. To investigate useful urinary biomarkers in UPJO children, the urinary proteome in UPJO infants is analyzed and it is compared with normal controls. A tandem mass tag (TMT)-based quantitative proteomics study is performed to analyze the proteome of bladder urine (BU) and pelvis urine (PU) from unilateral UPJO infants with differential renal function less than 40% and they are compared with normal control urine (CON). GO analysis is then utilized to analyze general characterization of the proteins. Proteomic results are verified by western blot. There are 81 and 186 proteins significantly changed in BU and PU groups, respectively, as compared to the CON group. Fifty proteins overlaps are found between these two sets of statistically significant differential proteins. These 50 common differential proteins are involved in multiple biological processes. The increased urinary abundance of Fetuin-A, AGP1, AGP2, Alpha-1-microglobulin/Bikunin Precursor (AMBP), and prostaglandin-H2D-isomerase (PGDS) are verified by western blot analysis. This proteomic analysis indicates that urinary Fetuin-A, AGP1, AGP2, protein AMBP, and PGDS may serve as noninvasive potential biomarkers and these proteins can help to further yield pathological mechanisms involved in UPJO.

Constant urinary proteins in healthy humans in a 520-day isolation experiment

The aim of this study was the search of permanent proteins of the urinary proteome during a 520-day isolation experiment at the Institute of Biomedical Problems (IBMP) Ground-Based Test Facility in controlled conditions, using an autonomous life support system. The object of the study was urine sampled from 6 normal male subjects aged 25 to 37. The biological material samples (the second morning urine fractions) were collected for proteomic investigations against the background, on the 50th, 93rd, 124th, 153rd, 180th, 251st, 274th, 303rd, 330th, 371st, 400th, and 427th days of isolation, and on the 7th day after its completion. The samples were analyzed using chromatography–mass spectrometry, while the obtained results were analyzed using bioinformatics resources. The following seven permanent proteins were identified and observed during the entire period of urine investigations: epidermal growth factor, polymeric immunoglobulin receptor, plasma serine protease inhibitor, Alpha 1 microglobulin/bikunin precursor (AMBP), keratin (type II cytoskeletal 1), collagen alpha-1 (VI) chain, and serum albumin.

Elevated levels of protein AMBP in cerebrospinal fluid of women with preeclampsia compared to normotensive pregnant women.

To investigate the cerebrospinal fluid (CSF) proteome of patients with preeclampsia (PE) and normotensive pregnant women, in order to provide a better understanding of brain involvement in PE. Ninety-eight CSF samples (43 women with PE and 55 normotensive controls) were analyzed by LC-MS/MS proteome profiling. CSF was obtained during the spinal puncture before caesarean delivery. Eight proteins were higher abundant and 17 proteins were lower abundant in patients with PE. The most significantly differentially abundant protein was protein AMBP (alpha-1-microglobulin/bikunin precursor). This finding was validated by performing an ELISA experiment (p = 0.002). The current study showed a clear difference between the protein profiles of CSF from patients with PE and normotensive pregnant women. Protein AMBP is a precursor of a heme-binding protein that counteracts the damaging effects of free hemoglobin, which may be related to the presence of free hemoglobin in CSF. Protein levels showed correlations with clinical symptoms during pregnancy and postpartum. To our knowledge, this is the first LC-MS/MS proteome profiling study on a unique set of CSF samples from (severe) preeclamptic patients and normotensive pregnant women.

Adsorption of Rotavirus, MS2 Bacteriophage and Surface-Modified Silica Nanoparticles to Hydrophobic Matter.

Adsorption to aquifer media is an important process in the removal of viruses from groundwater. Even though hydrophobic interactions have been shown to contribute to adsorption, little is known about the hydrophobicity of viruses found in groundwater. In this study, the hydrophobicity of rotavirus, MS2 bacteriophage and DNA-labelled silica nanoparticles (SiNPs) coated with glycoprotein, protein A and alpha-1-microglobulin/bikunin precursor(AMBP) was investigated. The hydrophobicity was experimentally determined by using a modified microbial adhesion to hydrocarbons (MATH) assay. The results were compared with the theoretical hydrophobicity of the viral capsid proteins and the proteins used to coat the nanoparticles, and with the results of adsorption tests with unmodified and organosilane-coated (hydrophobic) silica sand. While most theoretical protein hydrophobicity values were similar, the results of the MATH assay suggested fundamental differences in the hydrophobicity of the viruses and the SiNPs. MS2 was found to be highly hydrophobic as based on the MATH hydrophobicity and a significantly enhanced adsorption to hydrophobic sand, whereas rotavirus was relatively hydrophilic. The MATH assay revealed that protein-coating of SiNP introduced some degree of hydrophobicity to hydrophilic SiNPs, enabling them to more closely mimic viral hydrophobicity. Our study also demonstrated that the protein-coated SiNPs better mimicked rotavirus adsorption to sand media (coated or not coated with hydrophobic organic matter) than the MS2. This further supports previous findings that these surface-modified SiNPs are useful surrogates in mimicking rotavirus retention and transport in porous media.

High level of serum AMBP is associated with poor response to paclitaxel–capecitabine chemotherapy in advanced gastric cancer patients

Gastric cancer is one of the most common human cancers and ranks the second in the global cancer-related mortality. The clinical outcome of patients with advanced gastric cancer (AGC) is markedly dependent on their response to the chemotherapy. Paclitaxel plus capecitabine, as a first-line regimen, is widely administrated in AGC patients, but more than a half of the patients have a poor response, possibly due to their resistance to the treatment. Therefore, it is important to identify potential responders to improve the efficacy of the chemotherapy. In the present study, we used an isobaric tag approach for relative and absolute quantification combined with ESI-QUAD-TOF/MS to identify potential predictive biomarkers for the chemotherapy. We found 211 serum proteins, and confirmed 17 candidates that were differentially present in the progression of disease (PD) group and the partial response (PR) group to the treatment of paclitaxel plus capecitabine. In further validation of the 17 candidates in the set of 12 PD and 12 PR AGC patients, we identified a higher level of AMBP (Alpha-1-Microglobulin/Bikunin Precursor) in the sera of PD patients than of the PR patients assayed by ELISA (9.13 ± 0.45 vs. 8.11 ± 0.26 μg/mL, p = 0.06) and by the Western blotting (relative gray value 396.4 ± 39.1 vs. 275.0 ± 34.76, p = 0.03), respectively. The receiver operating characteristics curve showed 75% sensitivity and 75% specificity of AMBP in AGC patients treated with the chemotherapy. Our data indicated that the high level of serum AMBP could predict the poor response of the AGC patients treated with the paclitaxel-capecitabine chemotherapy, which could be used as a potential biomarker to identify patients who would benefit from this chemotherapeutic regimen.

Association and expression quantitative trait loci (eQTL) analysis of porcine AMBP, GC and PPP1R3B genes with meat quality traits

The aim of this research was to screen polymorphism and to perform association study of porcine AMBP (alpha-1-microglobulin/bikunin precursor), GC (group-specific component protein) and PPP1R3B (protein phosphatase 1, regulatory (inhibitor) subunit 3B) genes with meat quality traits as well as to unravel the transcriptional regulation of these genes by expression QTL (eQTL) study. For this purpose, Duroc×Pietrain F2 resource population (DuPi; n=313) and a commercial breed Pietrain (Pi; n=110) were used for association and only DuPi for expression and eQTL study. A SNP was identified in the genes AMBP (g.22229C>T), GC (g.398C>T) and PPP1R3B (c.479A>G), respectively. In DuPi SNP of AMBP was associated (P<0.05) with meat colour, pH(1L), pH(24L), pH(24H) and conductivity(24L); SNP of GC showed tendency to association (P<0.10) with pH24H, conductivity(1L) and thawing loss, and SNP of PPP1R3B was associated (P<0.05) with meat colour, pH(1L), pH(24L), pH(24H) and shear force. In Pi SNPs of AMBP and GC was associated with pH(24H) and PPP1R3B SNP was associated with pH(24L). The mRNA levels in Longissimus dorsi muscle tissue of these three genes were evaluated by using qRT-PCR to identify association between gene expression and meat quality traits as well as to analyse eQTL. The mRNA expression of PPP1R3B associated with pH(24L) (P<0.05). Expression of these three genes was higher in animals with low pH of muscle. Linkage analysis using QTL Express revealed ten trans-regulated eQTL on seven porcine autosomes. Suggestive eQTL [P<0.05, CW (chromosome-wide)] were found for PPP1R3B on SSC3 and 13. These results revealed that genetic variation and gene expression of these genes are associated with the meat quality traits. These three genes could influence meat quality and could be potential positional, physiological and functional candidate gene for meat quality traits in pigs. However, the analysis of eQTL also suggested that we need to consider additional genes encoding for transcription factors (TF), via fine-mapping underlying the eQTL peaks, in order to understand interaction among these genes.

Biomarker discovery in subclinical mycobacterial infections of cattle.

BackgroundBovine tuberculosis is a highly prevalent infectious disease of cattle worldwide; however, infection in the United States is limited to 0.01% of dairy herds. Thus detection of bovine TB is confounded by high background infection with M. avium subsp. paratuberculosis. The present study addresses variations in the circulating peptidome based on the pathogenesis of two biologically similar mycobacterial diseases of cattle.Methodology/Principal FindingsWe hypothesized that serum proteomes of animals in response to either M. bovis or M. paratuberculosis infection will display several commonalities and differences. Sera prospectively collected from animals experimentally infected with either M. bovis or M. paratuberculosis were analyzed using high-resolution proteomics approaches. iTRAQ, a liquid chromatography and tandem mass spectrometry approach, was used to simultaneously identify and quantify peptides from multiple infections and contemporaneous uninfected control groups. Four comparisons were performed: 1) M. bovis infection versus uninfected controls, 2) M. bovis versus M. paratuberculosis infection, 3) early, and 4) advanced M. paratuberculosis infection versus uninfected controls. One hundred and ten differentially elevated proteins (P≤0.05) were identified. Vitamin D binding protein precursor (DBP), alpha-1 acid glycoprotein, alpha-1B glycoprotein, fetuin, and serine proteinase inhibitor were identified in both infections. Transthyretin, retinol binding proteins, and cathelicidin were identified exclusively in M. paratuberculosis infection, while the serum levels of alpha-1-microglobulin/bikunin precursor (AMBP) protein, alpha-1 acid glycoprotein, fetuin, and alpha-1B glycoprotein were elevated exclusively in M. bovis infected animals.Conclusions/SignificanceThe discovery of these biomarkers has significant impact on the elucidation of pathogenesis of two mycobacterial diseases at the cellular and the molecular level and can be applied in the development of mycobacterium-specific diagnostic tools for the monitoring progression of disease, response to therapy, and/or vaccine based interventions.

Proteomic profiling of amniotic fluid in preterm labor using two-dimensional liquid separation and mass spectrometry

Objective. Simultaneous analysis of the protein composition of biological fluids is now possible. Such an approach can be used to identify biological markers of disease and to understand the pathophysiology of disorders that have eluded classification, diagnosis, and treatment. The purpose of this study was to analyze the differences in protein composition of the amniotic fluid of patients in preterm labor.Study design. Amniotic fluid was obtained by amniocentesis from three groups of women with preterm labor and intact membranes: (1) women without intra-amniotic infection/inflammation (IAI) who delivered at term, (2) women without IAI who delivered a preterm neonate, and (3) women with IAI. Intra-amniotic infection was defined as a positive amniotic fluid culture for microorganisms. Intra-amniotic inflammation was defined as an elevated amniotic fluid interleukin (IL)-6 (≥2.3 ng/mL). Two-dimensional (2D) chromatography was used for analysis. The first dimension separated proteins by isoelectric point, while the second, by the degree of hydrophobicity. 2D protein maps were generated using different experimental conditions (reducing agents as well as protein concentration). The maps were used to discern subsets of isoelectric point/hydrophobicity containing differentially expressed proteins. Protein identification of differentially expressed fractions was conducted with mass spectrometry. Enzyme-linked immunosorbent assays (ELISA) as well as surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS)-based on-chip antibody capture immunoassays were also used for confirmation of a specific protein that was differentially expressed.Results. (1) Amniotic fluid protein composition can be analyzed using a combination of 2D liquid chromatography and mass spectrometry for the identification of proteins differentially expressed in patients in preterm labor. (2) While total insulin-like growth factor-binding protein-1 (IGFBP-1) concentration did not change, IGFBP-1 fragments at about 13.5 kDa were present in patients with IAI. (3) Proteins that were over-expressed in group 1 included von Ebner gland protein precursor, IL-7 precursor, apolipoprotein A1, tropomyosin sk1 (TPMsk1) fragment, ribosomal protein S6 kinase alpha-3, and alpha-1-microglobulin/bikunin precursor (AMBP). (4) Proteins that were over-expressed in group 3 included fibrinopeptide B, transferrin, major histocompatibility complex (MHC) class 1 chain-related A antigen fragment, transcription elongation factor A, sex-determining region Y (SRY) box 5 protein, Down syndrome critical region 2 protein (DSCR2), and human peptide 8 (HP8). (5) One protein, retinol-binding protein, was over-expressed in women who delivered preterm, regardless of the presence of IAI.Conclusions. A combination of techniques involving 2D chromatography, mass spectrometry, and immunoassays allows identification of proteins that are differentially regulated in the amniotic fluid of patients with preterm labor. Specifically, the amount of the IGFBP-1 fragments at approximately 13.5 kDa was found to be increased in patients with IAI, while the amount of the intact form of IGFBP-1 was decreased.

Knockdown of alpha-1-microglobulin bikunin precursor (AMBP) causes ocular, and craniofacial defects

Knockdown of alpha-1-microglobulin bikunin precursor (AMBP) causes ocular, and craniofacial defects

The ORF3 Protein of Hepatitis E Virus Interacts with Liver-specific α1-Microglobulin and Its Precursor α1-Microglobulin/Bikunin Precursor (AMBP) and Expedites Their Export from the Hepatocyte

Hepatitis E virus (HEV), a plus-stranded RNA virus contains three open reading frames. Of these, ORF1 encodes the viral nonstructural polyprotein; ORF2 encodes the major capsid protein and ORF3 codes for a phosphoprotein of undefined function. Using the yeast two-hybrid system to screen a human cDNA liver library we have isolated, an N-terminal deleted protein, alpha(1) -microglobulin/bikunin precursor (AMBP) that specifically interacts with the ORF3 protein of HEV. Independently cloned, full-length AMBP was obtained and tested positive for interaction with ORF3 using a variety of in vivo and in vitro techniques. AMBP, a liver-specific precursor protein codes for two different unrelated proteins alpha(1)-microglobulin (alpha(1)m) and bikunin. alpha(1) m individually interacted with ORF3. The above findings were validated by COS-1 cell immunoprecipitation, His(6) pull-down experiments, and co-localization experiments followed by fluorescence resonance energy transfer analysis. Human liver cells showing co-localization of ORF3 with endogenously expressing alpha(1) m showed a distinct disappearance of the protein from the Golgi compartment, suggesting that ORF3 enhances the secretion of alpha(1)m out of the hepatocyte. Using drugs to block the secretory pathway, we showed that alpha m was not degraded in the presence of ORF3. Finally, (1)pulse labeling of alpha(1)m showed that its secretion was expedited out of the liver cell at faster rates in the presence of the ORF3 protein. Hence, ORF3 has a direct biological role in enhancing alpha(1)m export from the hepatocyte.

Assignment of the alpha-1-microglobulin bikunin precursor (AMBP) gene to porcine chromosome 1 via a new PCR-based polymorphism.

Assignment of the alpha-1-microglobulin bikunin precursor (AMBP) gene to porcine chromosome 1 via a new PCR-based polymorphism.

An array of binding sites for hepatocyte nuclear factor 4 of high and low affinities modulates the liver-specific enhancer for the human α1-microglobulin/bikunin precursor

Alpha1-Microglobulin and bikunin are two plasma glycoproteins encoded by a gene for alpha1-microglobulin/bikunin precursor (AMBP). The strict liver-specific transcription of the AMBP gene is controlled by an elaborate and remote enhancer made of six clustered boxes numbered 1 to 6 (core enhancer) that are binding sites for the hepatocyte-enriched nuclear factors HNF-1, HNF-4, HNF-3, HNF-1, HNF-3 and HNF-4 respectively. Three further boxes, 7 to 9, have now been found in the enhancer area in a position 5' of box 2, 5' of box 1 and 3' of box 6, respectively. Electrophoretic mobility-shift assays with nuclear extracts from the HepG2 hepatoma cell line demonstrated that boxes 7 and 8 are both functional HNF-4-binding sites of high and low affinity respectively, whereas no binding capacity of box 9 was detected by this method. Transfection of HepG2 and Chinese hamster ovary cells with chloramphenicol acetyltransferase constructs harbouring the core or extended AMBP enhancer with wild-type or mutated boxes and co-transfection with expression plasmids for a wild-type or defective HNF-4 identified box 7 as an essential element for the basal activity of this enhancer. The response of boxes 7 and 8 varies with the level of HNF-4 in cells. Box 9 exhibits a repressor activity that can be detected when box 8 is ablated. In vivo this corresponds to conditions of low box 8 occupancy when the intracellular level of HNF-4 is limited. These results reinforce the view that the AMBP enhancer is a quite elaborate and unusual example of a modular enhancer whose activity is fine-tuned by the level of cognate nuclear factors in the cell.

The three heavy-chain precursors for the inter-α-inhibitor family in mouse: new members of the multicopper oxidase protein group with differential transcription in liver and brain

The inter-alpha-inhibitor (I alpha I) family is comprised of the plasma protease inhibitors I alpha I, inter-alpha-like inhibitor (I alpha LI), pre-alpha-inhibitor (P alpha I) and bikunin. I alpha I, I alpha LI and P alpha I are distinct assemblies of bikunin with one of three heavy (H) chains designated H1, H2 and H3. These H chains and bikunin are respectively encoded by a set of three H genes and an alpha 1-microglobulin/bikunin precursor (AMBP) gene. All four gene products undergo maturation steps from precursor polypeptides. The full-length cDNAs for the H1-, H2- and H3-chain precursors were cloned from a mouse liver cDNA library and sequenced. Extensive searches of amino acid sequence similarities to other proteins in databanks revealed (i) a highly significant similarity of the C-terminal sequence in the three H-chain precursors to the multicopper-binding domain in the group of multicopper oxidase proteins and (ii) the presence of von Willebrand type-A domains in the mature H chains. Amino acid sequence comparisons between the three mouse H1-, H2- and H3-chain precursors and their human counterparts allowed us to appraise the timing and order of occurrence of the three H-chain genes from a shared ancestor during mammalian evolution. Owing to a multiple alignment of the six mouse and human nucleotide sequences for these H-chain precursors, a reverse transcriptase PCR assay with degenerate oligonucleotides was designed, allowing us to (i) present evidence that no mRNAs for further H genes exist in mouse liver and (ii) demonstrate a previously undescribed transcription of the H2- and H3-chain mRNAs in mouse brain, which contrasts with the expression of all four, H1, H2, H3 and AMBP, mRNAs in liver.

Hierarchy and positive/negative interplays of the hepatocyte nuclear factors HNF-1, -3 and -4 in the liver-specific enhancer for the human alpha-1-microglobulin/bikunin precursor.

Alpha-1-microglobulin and bikunin are two plasma glycoproteins encoded by an alpha-1-microglobulin/bikunin precursor (AMBP) gene. The strict liver-specific expression of the AMBP gene is controlled by a potent enhancer made of six clustered boxes numbered 1-6 that have been reported to be proven or potential binding sites for the hepatocyte-enriched nuclear factors HNF-1, -4, -3, -1, -3, -4, respectively. In the present study, electromobility shift assays of wild-type or mutated probes demonstrated that the boxes 1-5 have a binding capacity for their cognate HNF protein. Box 5 is also a target for another, as yet unidentified, factor. A functional analysis of the wild-type or mutated enhancer, driving its homologous promoter and a reporter CAT gene in the HepG2 hepatoma cell line, demonstrated that all six boxes participate in the enhancer activity, with the primary influence of box 4 (HNF-1) and box 2 (HNF-4). A similar analysis in the HNF-free CHO cell line co-transfected with one or several HNF factors further demonstrated various interplays between boxes: box 3 (HNF-3 alpha and beta) has a negative influence over the major HNF-4 box 2 as well as a positive influence over the major HNF-1 box 4.