Expression of the AMBP gene transcript and its two protein products,alpha(1)-microglobulin and bikunin,in mouse embryogenesis

The expression pattern of the alpha(1)-microglobulin/bikunin precursor (AMBP) gene, and its two protein products were studied in mouse embryos of 8.5-15.5 days of embryonic development by in situ hybridization and immunohistochemistry. AMBP mRNA is strongly transcribed in liver parenchyma, pancreas, and intestine epithelium. Sites of weaker expression are the vessels of the umbilical cord, the developing vertebral bodies, and kidney. The alpha(1)-microglobulin and bikunin proteins are accordingly present in developing hepatocytes, pancreas, kidney, and gut. However, additional sites of protein distribution were found that do not correlate to mRNA localization: alpha(1)-microglobulin was found in myocytes and bikunin in cardiac muscle, nervous system microvasculature, and connective tissue. Both proteins were found in brain mesenchyme and meninges. Thus, a restricted expression of the AMBP mRNA in a few organs contrasts to a widespread and unique distribution of each of the two proteins.     

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.     

Cloning of hibernation-related genes of bullfrog (Rana catesbeiana) by cDNA subtraction

Seven genes specifically expressed during hibernation in the bullfrog (Rana catesbeiana) were cloned from a subtracted cDNA library constructed from livers of winter bullfrogs. Those genes were fibrinogen alpha-subunit, fibrinogen gamma-subunit, complement component C3, alpha-1-microglobulin/bikunin precursor (AMBP), transferrin, apoferritin middle subunit and one novel gene. Northern hybridization has indicated that these seven genes were specifically induced or enhanced in winter. Above all, expression of the novel gene was specifically induced in winter in liver, though the expression of that was neither induced in bullfrog nor Xenopus laevis by cold treatment. The novel gene, which was designated as rc-hirp (Rana catesbeiana hibernation-related protein), encoded 420 base pairs length and a putative protein of 139 amino acid residues. Annual analyses of the expression of these genes have suggested that the seven winter-specific genes are playing an important role in hibernation processes.     

The ORF3 protein of hepatitis E virus interacts with liver-specific alpha1-microglobulin and its precursor alpha1-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.     

The 41-amino-acid C-terminal region of the hepatitis E virus ORF3 protein interacts with bikunin, a kunitz-type serine protease inhibitor

Hepatitis E virus (HEV), a human plus-stranded RNA virus, contains three open reading frames (ORF). Of these, ORF1 encodes the viral nonstructural polyprotein, ORF2 encodes the major capsid protein, and ORF3 codes for a phosphoprotein of undefined function. Recently, using the yeast two-hybrid system to screen a human cDNA liver library, we have isolated and characterized AMBP (alpha1-microglobulin/bikunin precursor), which specifically interacts with the ORF3 protein of HEV. The ORF3 protein expedites the processing and secretion of alpha1-microglobulin. When checked individually for interaction, the second processed protein from AMBP, bikunin, strongly interacted with the full-length ORF3 protein. This protein-protein interaction has been validated by immunoprecipitation in both COS-1 and Huh7 cells and by His6 pull-down assays. In dual-labeling immunofluorescent staining, followed by fluorescence microscopy of transfected human liver cells, ORF3 colocalized with endogenously expressed bikunin. Finally, a 41-amino-acid C-terminal region of ORF3 has been found to be responsible for interacting with bikunin. The importance of this virus-host protein-protein interaction, with reference to the viral life cycle, has been discussed.     

Homologous chromosomal locations of the four genes for inter-alpha-inhibitor and pre-alpha-inhibitor family in human and mouse: assignment of the ancestral gene for the lipocalin superfamily.

The inter-alpha-inhibitor (I alpha I) and pre-alpha-inhibitor (P alpha I) family is composed of three plasma protease inhibitors, I alpha I, P alpha I, and bikunin, whose chains are encoded by a set of three evolutionarily related heavy (H) chain genes designated H1, H2, and H3 and a fourth gene, the so-called alpha 1-microglobulin/bikunin precursor (AMBP) gene. The latter codes for a precursor that splits into: (i) alpha 1-microglobulin, which belongs to the lipocalin superfamily; and (ii) bikunin, which is made up of two tandemly arranged protease inhibitor domains and belongs to the superfamily of Kunitz-type protease inhibitors. The bikunin chain is found in I alpha I and P alpha I molecules and it is also present as a free molecule in plasma. In human, the AMBP and H2 genes have been mapped to 9q32-q34 and 10p14-p15, respectively, while the H1 and H3 genes are tandemly located at 3p21.1-p21.2. In situ hybridization mappings indicate that the mouse AMBP gene (Intin-4) is located at 4C1----C4, and the H1 (Intin-1) and H3 (Intin-3) genes are colocated at 14A2----C1. In interspecific backcrosses (C57BL/6Pas x Mus spretus) a TaqI restriction variant in (and/or near) the H2 (Intin-2) gene identified a linkage of this gene with other polymorphic loci, which assigns Intin-2 to the centromeric area of chromosome 2. All such assignments are in conserved chromosomal regions between human and mouse. Therefore the genetic events that gave rise to the four I alpha I family genes took place prior to the divergence between human and mouse.(ABSTRACT TRUNCATED AT 250 WORDS)     

Biosynthesis of bikunin (urinary trypsin inhibitor) in rat hepatocytes.

One of the major sulfated proteins secreted by rat hepatocytes contains a low-sulfated chondroitin sulfate chain and its apparent molecular mass upon sodium dodecyl sulfate/polyacrylamide gel electrophoresis shifts from 40 to 28 kDa upon chondroitinase ABC treatment (E. M. Sj?berg and E. Fries, 1990, Biochem. J. 272, 113-118). These properties suggest that this protein is the rat homologue of the major trypsin inhibitor of human urine which was recently named bikunin. In serum, bikunin occurs mainly as a subunit of the pre-alpha-inhibitor and the inter-alpha-inhibitor; in these proteins it is covalently linked to the other polypeptides through its chondroitin sulfate chain. Bikunin has been shown to be synthesized by liver cells as a 42-kDa precursor, in which it is linked to alpha 1-microglobulin by two basic amino acids. We have isolated bikunin from rat urine and prepared antibodies against it. In rat hepatocytes pulse-labeled with [35S]methionine, these antibodies precipitated a labeled protein of 42 kDa. Upon chase, three different labeled proteins were recognized by the antibodies in the medium: one protein of 40 kDa (free bikunin), one of 125 kDa (presumably pre-alpha-inhibitor), and one greater than 240 kDa (possibly a protein related to the inter-alpha-inhibitor). Pulse-chase experiments with [35S]sulfate showed that these proteins occurred intracellularly as precursors containing alpha 1-microglobulin. These results demonstrate that the completion of the chondroitin sulfate chain and its coupling to other polypeptide chains occur before the cleavage of the alpha 1-microglobulin/bikunin precursor.     

Biomarker Discovery in Subclinical Mycobacterial Infections of Cattle

Background

Bovine 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 Findings

We 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/Significance

The 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.     

Heavy Chain Transfer by Tumor Necrosis Factor-stimulated Gene 6 to the Bikunin Proteoglycan

We present data that hyaluronan (HA) polysaccharides, about 14–86 monosaccharides in length, are capable of accepting only a single heavy chain (HC) from inter-α-inhibitor via transfer by tumor necrosis factor-stimulated gene 6 (TSG-6) and that this transfer is irreversible. We propose that either the sulfate groups (or the sulfation pattern) at the reducing end of the chondroitin sulfate (CS) chain of bikunin, or the core protein itself, enables the bikunin proteoglycan (PG) to accept more than a single HC and permits TSG-6 to transfer these HCs from its relatively small CS chain to HA. To test these hypotheses, we investigated HC transfer to the intact CS chain of the bikunin PG, and to the free chain of bikunin. We observed that both the free CS chain and the intact bikunin PG were only able to accept a single HC from inter-α-inhibitor via transfer by TSG-6 and that HCs could be swapped from the bikunin PG and its free CS chain to HA. Furthermore, a significant portion of the bikunin PG was unable to accept a single heavy chain. We discuss explanations for these observations, including the intracellular assembly of inter-α-inhibitor. In summary, these data demonstrate that the sulfation of the CS chain of bikunin and/or its core protein promote HC transfer by TSG-6 to its relatively short CS chain, although they are insufficient to enable the CS chain of bikunin to accept more than one HC in the absence of other cofactors.     

The alpha1-microglobulin/bikunin gene: characterization in mouse and evolution.

The 129Sv mouse gene coding for the alpha1-microglobulin/bikunin precursor has been isolated and characterized. The 11kb long gene contains ten exons, including six 5'-exons coding for alpha1-microglobulin and four 3'-exons encoding bikunin. Exon 7 also codes for the tribasic tetrapeptide RARR which connects the alpha1-microglobulin and bikunin parts. The sixth intron, which separates the alpha1-microglobulin and bikunin encoding parts, was compared in the human, mouse and a fish (plaice) gene. The size of this intron varies considerably, 6.5, 3.3 and 0.1kb in man, mouse and plaice, respectively. In all three genes, this intron contains A/T-rich regions, and retroposon elements are found in the first two genes. This indicates that this sixth intron is an unstable region and a hotspot for recombinational events, supporting the concept that the alpha1-microglobulin and bikunin parts of this gene are assembled from two ancestral genes. Finally, the nonsynonymous nucleotide substitution rate of the gene was determined by comparing coding sequences from ten vertebrate species. The results indicate that the alpha1-microglobulin part of the gene has evolved faster than the bikunin part.     

alpha(1)-Microglobulin: a yellow-brown lipocalin

alpha(1)-Microglobulin, also called protein HC, is a lipocalin with immunosuppressive properties. The protein has been found in a number of vertebrate species including frogs and fish. This review summarizes the present knowledge of its structure, biosynthesis, tissue distribution and immunoregulatory properties. alpha(1)-Microglobulin has a yellow-brown color and is size and charge heterogeneous. This is caused by an array of small chromophore prosthetic groups, attached to amino acid residues at the entrance of the lipocalin pocket. A gene in the lipocalin cluster encodes alpha(1)-microglobulin together with a Kunitz-type proteinase inhibitor, bikunin. The gene is translated into the alpha(1)-microglobulin-bikunin precursor, which is subsequently cleaved and the two proteins secreted to the blood separately. alpha(1)-Microglobulin is found in blood and in connective tissue in most organs. It is most abundant at interfaces between the cells of the body and the environment, such as in lungs, intestine, kidneys and placenta. alpha(1)-Microglobulin inhibits immunological functions of white blood cells in vitro, and its distribution is consistent with an anti-inflammatory and protective role in vivo.     

Expression of a functional proteinase inhibitor capable of accepting xylose: bikunin

Bikunin is a Kunitz-type proteinase inhibitor, which is cross-linked to heavy chains via a chondroitin sulfate chain, forming inter-alpha-inhibitor and related molecules. Rat bikunin was produced by baculovirus-infected insect cells. The protein could be purified with a total yield of 20 mg/liter medium. Unlike naturally occuring bikunin the recombinant protein had no galactosaminoglycan chain. Endoglycosidase digestion also suggested that the recombinant form lacked N-linked oligosaccharides. Bikunin is translated as a part of a precursor, alpha1-microglobulin/bikunin, but the functional significance of the cotranslation is unknown. Our results indicate that the proteinase inhibitory function of bikunin is not regulated by the alpha1-microglobulin-part of the alpha1-microglobulin/bikunin precursor since recombinant bikunin had the same trypsin inhibitory activity as the recombinant precursor. Both free bikunin and the precursor were also functional as a substrate in an in vitro xylosylation system. This demonstrates that the alpha1-microglobulin-part is not necessary for the first step of galactosaminoglycan assembly.     

The Role of Amino-Terminal and Carboxy-Terminal Extensions in the Processing and Translocation of a Plant Proteinase (Actinidin) Expressed in the Yeast Saccharomyces cerevisiae

Summary A plant proteinase gene naturally occuring in the Kiwi fruit plant (Actinidia chinensis) has been expressed in a yeast Saccharomyces cerevisiae. Different gene constructions consisting of different portions of the whole actinidin-encoding gene have been created and expressed using an expression-secretion yeast vector. It was observed that the amino- and carboxy-terminal extensions of the actinidin-encoding gene were required for the correct expression of the gene in yeast. A gene construction lacking both amino- and C-terminal extensions did not result in a detectable protein product. Similarly, a gene construction consisting of the amino-terminal extension plus mature actinidin-encoding DNA did not result in a detectable expression. However, intracellular expression was observed when a gene construction consisting of mature actinidin-encoding DNA plus C-terminal extension portion was employed. The expressed polypeptide was found however not to be correctly processed as it had a bigger size than the native actinidin. The correctly processed polypeptide was expressed intracellularly when the full-length actinidin cDNA was expressed in a vacuolar protease-proficient yeast strain. However, when a vacuolar protease-deficient yeast strain was employed, it was found that the precursor protein was not correctly processed, suggesting that the actinidin precursor had entered the vacuole and undergone proteolytic processing. The full-length actinidin cDNA consisted of the amino-terminal extension DNA, mature actinidin-encoding DNA, and C-terminal extension DNA. The results thus suggested that both amino- and C-terminal extensions were required for correct expression and processing of actinidin in yeast. The intracellular expression also suggested that the actinidin-encoding sequences contain intracellular targeting sequences which override the secretion signal included in the expression-secretion vector.     

Genetic Control of L-a and L-(Bc) Dsrna Copy Number in Killer Systems of SACCHAROMYCES CEREVISIAE

M dsRNA in yeast encodes a toxin precursor and immunity protein, whereas L-A dsRNA encodes the 81,000-dalton major protein of the intracellular particles in which both L-A and M are found. L-(BC) dsRNA(s) are found in particles with different coat proteins. We find that M dsRNA lowers the copy number of L-A, but not L-(BC). The SKI gene products lower the copy number of L-(BC), L-A, M₁ and M₂. This is the first known interaction of L-(BC) with any element of the killer systems. The MAK3, MAK10 and PET18 gene products are necessary for L-A maintenance and replication, but mutations in these genes do not affect L-(BC) copy number. Mutations in MAK1, MAK4, MAK7, MAK17 and MAK24 do not detectably affect copy number of L-(BC) or L-A.     

Jun NH2-terminal kinase (JNK) interacting protein 1 (JIP1) binds the cytoplasmic domain of the Alzheimer's beta-amyloid precursor protein (APP).

The familial Alzheimer's disease gene product amyloid beta precursor protein (APP) is sequentially processed by beta- and gamma-secretases to generate the Abeta peptide. The biochemical pathway leading to Abeta formation has been extensively studied since extracellular aggregates of Abeta peptides are considered the culprit of Alzheimer's disease. Aside from its pathological relevance, the biological role of APP processing is unknown. Cleavage of APP by gamma-secretase releases, together with Abeta, a COOH-terminal APP intracellular domain, termed AID. This peptide has recently been identified in brain tissue of normal control and patients with sporadic Alzheimer's disease. We have previously shown that AID acts as a positive regulator of apoptosis. Nevertheless, the molecular mechanism by which AID regulates this process remains unknown. Hoping to gain clues about the function of APP, we used the yeast two-hybrid system to identify interaction between the AID region of APP and JNK-interacting protein-1 (JIP1). This molecular interaction is confirmed in vitro, in vivo by fluorescence resonance energy transfer (FRET), and in mouse brain lysates. These data provide a link between APP and its processing by gamma-secretase, and stress kinase signaling pathways. These pathways are known regulators of apoptosis and may be involved in the pathogenesis of Alzheimer's disease.