Bovine alpha 1-microglobulin/bikunin. Isolation and characterization of liver cDNA and urinary alpha 1-microglobulin

cDNA coding for alpha 1-microglobulin, an immunoregulatory plasmaprotein, was isolated from bovine liver. The sequence of a total of 1258 nucleotides revealed an open reading frame of 352 amino acids. This included alpha 1-microglobulin, 182 amino acids, and bikunin, the light chain of the plasmaprotein inter-alpha-inhibitor, 147 amino acids. The two proteins were connected by a basic tetrapeptide, R-A-R-R, which conforms to the consensus sequence recognized by endoproteolytic cleavage enzymes. The deduced amino acid sequence showed a high degree of identity with alpha 1-microglobulin and bikunin sequences from other species, and the alpha 1-microglobulin part displayed sequence motifs typical for members of the lipocalin protein superfamily. A single alpha 1-microglobulin/bikunin mRNA with a size of around 1300 nt was found in bovine liver. The mature alpha 1-microglobulin protein was isolated from bovine urine, and partly characterized. It was found to be a globular molecule with an apparent molecular weight of 23,300, containing one N-linked and at least on O-linked oligosaccharide, one intra-chain disulfide bridge and an electrophoretic heterogeniety with a pI-value of 4.1-5.2

Expression of rat alpha 1-microglobulin-bikunin in baculovirus-transformed insect cells

cDNA encoding rat alpha 1-microglobulin-bikunin was ligated into the transfer vector pVL 1392 and recombined with a wild-type baculovirus. The resulting alpha 1-microglobulin-bikunin-encoding baculovirus was used to infect Trichoplusia ni (Hi-5) insect cells. The infected cells secreted alpha 1-microglobulin with maximal concentrations of 15 mg/liter 5 days after infection. The secreted proteins migrated upon SDS-PAGE as two major protein bands, 40 and 26 kDa, corresponding to alpha 1-microglobulin-bikunin and free alpha 1-microglobulin. The results suggested that the cells secreted mostly alpha 1-microglobulin-bikunin, which subsequently was cleaved in the medium, yielding free alpha 1-microglobulin. Both forms were isolated by monoclonal anti-alpha 1-microglobulin affinity chromatography, and alpha 1-microglobulin-bikunin separated from free alpha 1-microglobulin by gel chromatography. The yields of purified alpha 1-microglobulin-bikunin and free alpha 1-microglobulin were approximately 1 and 5 mg, respectively, per liter medium. Insect cell alpha 1-microglobulin displayed a size, shape, and charge heterogeneity similar to alpha 1-microglobulin isolated from rat urine. A panel of monoclonal antibodies raised against urinary alpha 1-microglobulin from several different species bound to rat urinary alpha 1-microglobulin and insect cell secreted alpha 1-microglobulin-bikunin and free alpha 1-microglobulin with approximately the same strength, indicating that the three proteins are folded in similar ways. The results of glycosidase treatments and lectin blotting indicate the absence of neuraminic acid but the presence of one N-linked oligosaccharide and an unspecified number of O-linked oligosaccharides in alpha 1-microglobulin-bikunin and free alpha 1-microglobulin

Receptor for IgA in group A streptococci : cloning of the gene and characterization of the protein expressed in Escherichia coli

The gene for an IgA-binding protein from a group A streptococcal strain was cloned and expressed in Escherichia coli. The IgA-binding protein, called protein Arp, was purified on IgA-Sepharose, allowing complete purification in a single step. Analysis of protein Arp by Western immunoblotting demonstrated a major IgA-binding band, with an apparent molecular weight of 42 kD. The purified protein was shown to bind serum IgA and secretory IgA, as well as monoclonal IgA of both subclasses. There was no binding to IgM, IgD or IgE, but a weak binding to IgG. Inhibition experiments with whole bacteria indicated that IgA and IgG bind at separate sites. Experiments with immunoglobulin fragments showed that protein Arp binds to the Fc region of both IgA and IgG. The equilibrium constant of the reaction between protein Arp and polyclonal human IgA was determined to be 5.6 x 10(8) M-1. Amino acid sequencing of protein Arp demonstrated a direct repeat of 7 amino acids in the NH2-terminal region, a feature previously found in several streptococcal M proteins. This suggests that protein Arp, like M proteins, may be a streptococcal virulence factor

An intriguing member of the lipocalin protein family : alpha 1-microglobulin

The plasma protein alpha 1-microglobulin is a member of the lipocalin protein superfamily. In the last few years, the work on alpha 1-microglobulin has given unexpected and promising new results. Of particular interest are its molecular association with immunoglobulin A and with proteinase inhibitors, and its interactions with the immune system

Cross-reacting monoclonal anti-alpha 1-microglobulin antibodies produced by multi-species immunization and using protein G for the screening assay

In order to generate monoclonal antibodies (MAb) directed against the low molecular weight glycoprotein alpha 1-microglobulin, a BALB/c mouse was immunized with a mixture of human, guinea pig, rat and rabbit alpha 1-microglobulin homologues (multi-species immunization) and boosted several times. On day 194, the mouse splenocytes were fused to SP2/0 myeloma cells. The resulting hybridomas were screened for anti-alpha 1-microglobulin activity against the alpha 1-microglobulin mixture or against the individual homologues. For this screening, protein G (the newly described IgG-binding streptococcal protein) was used in a solid-phase radioimmunoassay. The binding of protein G to immobilized antigen-antibody complexes was enhanced by pre-incubation with rabbit anti-mouse immunoglobulin G. The result was a panel of nine established hybridoma lines, all producing unique monoclonal antibodies, of IgG1 or IgG2a class, to alpha 1-microglobulin. The antibodies were not only reactive in solid-phase radioimmunoassay, but they could also immunoprecipitate 125I-labeled soluble alpha 1-microglobulin. Moreover, they reacted specifically with the alpha 1-microglobulin band in Western blots of urinary proteins separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Such monoclonal antibodies are potentially valuable reagents for the further characterization of alpha 1-microglobulin

Detection and purification of rat and goat immunoglobulin G antibodies using protein G-based solid-phase radioimmunoassays

Using the newly described streptococcal surface protein, protein G, which has powerful immunoglobulin G binding properties, solid-phase radioimmunoassays were developed for the quantitation of goat and rat immunoglobulin G bound to the plastic surface of microtiter plates. The binding of goat immunoglobulin G to the surface via a specific antigen (guinea pig alpha 1-microglobulin) permitted the determination of antigen-specific antibodies with a detection limit of 50-100 ng. Optimum assay conditions were established and the whole assay procedure could be brought to completion at room temperature in less than a working day. The value of the assays was illustrated by monitoring rat and goat immunoglobulin G antibodies during their purification from whole sera by classical chromatographic procedures

Enzyme linked immunosorbent assay using alkaline phosphatase conjugated with streptococcal protein G

Protein G, an IgG-binding protein, purified from the surface of group G streptococci, was coupled to alkaline phosphatase. The conjugate was used for detection of polyclonal goat and rabbit antibodies and monoclonal mouse IgG1, IgG2a and IgG2b in an enzyme-linked immunosorbent assay. A two-step coupling procedure was used, in which glutaraldehyde was allowed to react with the enzyme, excess glutaraldehyde was then removed by dialysis, and finally protein G added to the glutaraldehyde-activated and polymerized alkaline phosphatase. The activity and yield of the conjugates were then tested in an enzyme-linked immunosorbent assay. Coupling of 25 micrograms protein G to 5 mg alkaline phosphatase gave a conjugate which could be used for more than 10,000 determinations with maximal antibody binding giving an absorbance of 2.0. Under these conditions, there was no need for separation of the reactants before using the protein G-alkaline phosphatase complex