The liver-specific human alpha(1)-microglobulin/bikunin precursor (AMBP) is capable of self-association

alpha-1-Microglobulin (A1M) and bikunin are two plasma glycoproteins encoded by an alpha-1-microglobulin/bikunin precursor (AMBP) gene. Despite their lack of any structural or functional relationship, both A1M and bikunin originate from AMBP cleavage by a furin-like protease that releases the two mature molecules. The AMBP gene maintains a tight control over its expression by a unique enhancer, which is controlled by several hepatocyte-enriched nuclear factors; however, the mechanisms of regulation of the intracellular levels of the AMBP protein are currently unknown. We report the ability of the AMBP protein to self-associate and form a dimer in a yeast environment using the yeast two-hybrid system and an in vitro dimerization assay. We also show that the A1M protein binds to its precursor protein, AMBP, whereas bikunin does not. This observation warrants further investigations for a dimerization-dependent intracellular control that AMBP may be involved in. The relevance of AMBP dimerization and its possible biological significance are postulated.     

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 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.     

Expression of fascin-1, the gene encoding the actin-bundling protein fascin-1, during mouse embryogenesis

Fascin-1 is an actin-bundling protein that contributes to the architecture and function of cell protrusions and microfilaments in cell adhesion, interactions and motility. Fascin-1 has been studied in cultured cells and by biophysical methods, but little is known about its distribution and functions in vertebrate development. As a first step to understanding the role of fascin-1 in embryogenesis, we have characterised the expression pattern of fascin-1 by in situ hybridisation on whole-mount and sectioned mouse embryos from embryonic day (E)8.0-E16.5. Fascin-1 was widely expressed throughout the embryo and the developing nervous system and mesenchymal tissues represented major sites of expression. Intense signals were observed in different regions of the brain, in the spinal cord and retina, and the cranial and dorsal root ganglia (DRG) appeared strongly positive. This neural expression remained strong throughout development. Fascin-1 was also present in the developing somites. High expression was detected in branchial arches and limb bud mesenchyme. At later stages, fascin-1 was expressed in different muscles of the face, skeletal muscles of the body, and in smooth muscle layers of several organs. Limb tendons appeared strongly positive. There was weak expression in heart ventricles. These results show that fascin-1 is principally expressed in neural and mesenchymal derivatives during embryonic development.     

SAXS studies of human protein HC (alpha1-microglobulin)

Protein HC is a low molecular weight heterogeneous glycoprotein widely distributed in human body fluids and belonging to the lipocalin superfamily. The monomer contains a single (183 amino acid residues long) peptide chain with 3 cysteine residues (2 of which form a disulfide bridge) and is glycosylated. The molecular mass of the glycosylated protein is about 27 kDa. Native gel electrophoresis results revealed partial oligomerisation of protein HC, which therefore was analysed by gel filtration. Two forms (monomer and dimer) of the protein HC were isolated. The SAXS data were recorded on an X33 camera using synchrotron radiation (lambda=0.15 nm) at X33 beamline at the DORIS storage ring of DESY (Hamburg, Germany). Solution scattering results permitted determination of the structural parameters of both forms of the protein studied. The monomer of protein HC is characterised by a radius of gyration R(G)= 2.20 nm and D(max)=6.3 nm and the dimer by R(G)=2.99 nm and D(max)=9.5 nm.     

Complementary DNA and deduced amino acid sequences of procine alpha 1-microglobulin and bikunin.

Analysis of complementary DNA for porcine alpha 1-microglobulin and bikunin indicates that both proteins result from proteolytic processing of a common precursor similar to that found in man. Complete primary structures of these proteins are deduced from the nucleic acid sequence and partially confirmed by peptide sequencing.     

Expression of the protein kinase D (PKD) family during mouse embryogenesis

The serine/threonine protein kinase D (PKD) family comprises of three members, PKD1 (PKCmu), PKD2 and PKD3 (PKCnu). Like the related C-type protein kinases (PKCs), PKDs are activated by diacylglycerol (DAG). PKDs have been implicated in numerous intracellular signaling pathways including vesicular transport, cell proliferation, survival, migration and immune responses. While experimental data on this recently discovered kinase family are starting to accumulate family member specific information is still sparse and only small effort has been taken to functionally differentiate the three PKDs. To address this issue we followed the expression patterns of PKD1, 2 and 3 during the development of the mouse embryo. Using specific probe sets for RT-PCR and in situ hybridization, we demonstrate shared and differential expression domains for the three PKD family members in both neuronal and non-neuronal tissues.     

Expression of the homeobox gene,Hox 2.1, during mouse embryogenesis

This article reviews recent studies on the expression of the homeobox gene, Hox 2.1, during mouse embryogenesis, using the technique of in situ hybridization. Differential hybridization of radiolabelled antisense versus sense strand RNA is first clearly detected in sections of 8.5 day post coitum (p.c.) early somite embryos. At 12.5 days p.c., higher levels of Hox 2.1 expression are seen in the spinal cord, extending into the base of the hind brain. Hybridization of antisense Hox 2.1 RNA is also seen in the spinal ganglia, in the nodose ganglia of the Xth cranial nerve (which contains derivatives of the neural crest arising from the posterior hind brain), and in the myenteric plexus. Mesodermal cells of certain visceral organs also express Hox 2.1 RNA, in particular the mesoderm of the lung, stomach and meso- and meta-nephric kidney. Comparison of the spatial domains of expression of mouse homeobox genes reveals a pattern consistent with the idea that they play a role in anteroposterior positional specification during embryogenesis.     

Expression of estrogen receptor alpha and beta during mouse embryogenesis.

In adult mammals numerous target tissues and organs for estrogens exist. Little is known about possible target organs during embryogenesis other than the reproductive tract and the gonads. This is the first report on the expression of estrogen receptor beta (ERbeta) in comparison with ERalpha mRNA during mouse embryogenesis. We found expression of estrogen receptor mRNA in the reproductive tract, but also in the atrial wall, brain, kidney, urethra, bladder neck, mammary gland primordium, midgut, cartilage primordia and perichondria.     

Expression of MUL, a gene encoding a novel RBCC family ring-finger protein, in human and mouse embryogenesis

In mammals, the SWI/SNF complex is involved in chromatin remodelling in a wide range of cellular events for which regulatory factors require access to DNA. In the present study, we analyzed in early postimplantation mouse embryos the expression pattern of BRM (SNF2alpha) and BRG1 (SNF2beta), which are both ATPase subunits of this complex. Contrarily to the previous studies conducted in adult mice, showing the ubiquitous and overlapping expressions of BRM and BRG1, we show that BRM expression is restricted to mesodermal tissues involved in early vasculogenesis and heart morphogenesis.     

Differential expression of two exons of the alpha1(XI) collagen gene (Col11a1) in the mouse embryo.

The amino terminal domain of collagen XI has a unique structure, which is believed to participate in the regulation of matrix assembly. Interestingly, several distinct isoforms of the amino terminal domain of alpha1(XI) and alpha2(XI) collagen chains exist as a result of alternative splicing. Here we report the analysis of the alternative splicing pattern of the mouse alpha1(XI) collagen gene (Col11a1). Like other vertebrate species, the mutually exclusive expression of exons 6A and 6B of Col11a1 results in the inclusion in the alpha1 chain of either an acidic peptide (pI 3.14) or a basic peptide (pI 11.66). Expression of these two exons was monitored in several tissues of the 16.5-day mouse embryo by in situ hybridization and immunohistochemistry, with exon-specific cDNA probes and peptide-specific antibodies, respectively. The results documented that isoforms containing the exon 6B-encoded peptide accumulate predominantly in the vertebrae, skeletal muscles and intestinal epithelium. By contrast, exon 6A products were found to be most abundant in the smooth muscle cells of the intestine, aorta and lung. The results using in situ hybridization confirmed those using immunohistochemistry. Albeit correlative, the evidence suggests distinct contributions of the two peptides to the differential assembly of tissue-specific matrices.     

Expression of stress inducible protein 1 (Stip1) in the mouse testis

Phthalate esters are considered endocrine disruptors that interfere with the endocrine balance and development of the mammalian testis. Mono-2-ethylhexyl phthalate (MEHP), the active metabolite of the ubiquitously used plasticizer di-2-ethylhexyl phthalate (DEHP), acts upon Sertoli cells as initial target. By subtractive cDNA libraries we identified genes deregulated as response to MEHP in primary cultures of mouse Sertoli cells. The expression of mouse stress inducible protein 1 (Stip1) was detected as upregulated as a result of MEHP exposure. Stip1 is a cochaperone protein that is homologous to the human heat shock cognate protein 70 (hsc70)/heat shock protein 90 (hsp90)-organizing protein (Hop). To assess the presence and localization of Stip1 in mouse testis and its potential role in stress defense, we studied the expression pattern of the Stip1 protein by immunohistochemistry and of the mRNA by in situ hybridization. Both the protein and the mRNA of Stip1 were mainly found in the cytoplasm of all types of spermatogonia and spermatocytes up till zygotene, the expression decreased during late pachytene and was very weak in diplotene spermatocytes and round spermatids. Interestingly, this expression pattern resembled the pattern of stress sensitivity of spermatogenic cells in that the most sensitive cell types show the weakest expression of Stip1. This suggests an important role for Stip1 in the ability of germ cells to survive in stress conditions including high temperatures.     

Expression specificity of the mouse exonuclease 1 (mExo1) gene.

Genetic recombination involves either the homo-logous exchange of nearly identical chromosome regions or the direct alignment, annealing and ligation of processed DNA ends. These mechanisms are involved in repairing potentially lethal or mutagenic DNA damage and generating genetic diversity within the meiotic cell population and antibody repertoire. We report here the identification of a mouse gene, termed mExo1 for mouse exonuclease 1, which encodes a approximately 92 kDa protein that shares homology to proteins of the RAD2 nuclease family, most notably human 5' to 3' exonuclease Hex1/hExo1, yeast exonuclease 1 (Exo1) proteins and Drosophila melanogaster Tosca. The mExo1 gene maps to distal chromosome 1, consistent with the recent mapping of the orthologous HEX1 / hEXO1 gene to chromosome 1q42-q43. mExo1 is expressed prominently in testis, an area of active homologous recombination, and spleen, a prominent lymphoid tissue. An increased level of mExo1 mRNA was observed during a stage of testis development where cells that are actively involved in meiotic recombination arise first and represent a significant proportion of the germ cell population. Comparative evaluation of the expression patterns of the human and mouse genes, combined with previous biochemical and yeast genetic studies, indicate that the Exo1-like proteins are important contributors to chromosome processing during mammalian DNA repair and recombination.     

Expression of the Lingo/LERN gene family during mouse embryogenesis

We have analysed the expression during mouse development of the four member Lingo/LERN gene family which encodes type 1 transmembrane proteins containing 12 extracellular leucine rich repeats, an immunoglobulin C2 domain and a short intracellular tail. Each family member has a distinct pattern of expression in the mouse embryo as is the case for the related NLRR, FLRT and LRRTM gene families. Lingo1/LERN1 is expressed in the developing trigeminal, facio-acoustic and dorsal root ganglia. An interesting expression pattern is also observed in the somites with expression localising to the inner surface of the dermomyotome in the ventro-caudal lip. Further expression is seen in lateral cells of the hindbrain and midbrain, lateral cells in the motor horn of the neural tube, the otic vesicle epithelium and epithelium associated with the developing gut. Lingo3/LERN2 is expressed in a broad but specific pattern in many tissues across the embryo. Lingo2/LERN3 is seen in a population of cells lying adjacent to the epithelial lining of the olfactory pit while Lingo4/LERN4 is expressed in the neural tube in a subset of progenitors adjacent to the motor neurons. Expression of all Lingo/LERN genes increases as the embryo develops but is low in the adult with only Lingo1/LERN1 and Lingo2/LERN3 being detectable in adult brain.     

Expression of the Xenopus GTP-binding protein gene Ran during embryogenesis

The Ran gene family encodes small GTP binding proteins that are associated with a variety of nuclear processes. We isolated a Xenopus Ran cDNA and analyzed the pattern of expression of this gene during embryogenesis. Ran is expressed maternally and later in the CNS, neural crest, mesenchyme, eyes, and otic vesicles. However, expression is not detected in the somites or the notochord. Received: 22 November 1999 / Accepted: 22 December 1999