Platelet-derived growth factor.

Platelet-derived growth factor (PDGF) is a potent activator for cells of mesenchymal origin. Two different PDGF chains termed A and B encoded by different genes have been identified leading to three different PDGF isoforms, the AA and BB homodimers and the AB heterodimer. All three forms have been observed in vivo and possess biological activity in vitro with the AA homodimer being the poorest cellular mitogen. The availability of highly purified recombinant PDGF isoforms was the initial basis for comparative studies in order to specify the different spectra of activity of the various PDGF species. This review is particularly focused on AB heterodimer as from the standpoint of heterologous gene expression, this species is the one with the highest demands concerning expression and purification protocols. This explains the fact that, in comparison to PDGF-BB, only very limited data on the in vivo application of PDGF-AB are available so far.

Translational regulation of a recombinant operon containing human platelet-derived growth factor (PDGF)-encoding genes in Escherichia coli: genetic titration of the peptide chains of the heterodimer AB.

A new strategy is described for the production of recombinant heteromultimeric proteins using Escherichia coli as host. A recombinant operon was constructed containing modified cDNA sequences encoding the mature A and B chains of human platelet-derived growth factor (PDGF). The relative expression rates of the PDGF genes were varied over a range equivalent to A:B ratios from 0.8 to 3.7 by means of translational regulation. This was achieved using two different translational initiation sequences (TIS) upstream from the respective coding regions, one derived from the E. coli atpE translational initiation region, and the other containing a sequence with extended complementarity to the 3' end of the 16S rRNA. The generation of mature PDGF A and B chains in different relative amounts in E. coli provided the basis for developing a novel procedure for the production of the biologically active PDGF heterodimer AB in large quantities. The general strategy is applicable to the preparation of a wide range of heteromultimeric complexes. Moreover, the described PDGF operon constitutes a compact and versatile model system for studies of the posttranscriptional regulation of gene expression.

On the structure of platelet-derived growth factor AA: C-terminal processing, epitopes, and characterization of cysteine residues.

The complete amino acid sequence analysis of the "short" form of rPDGF-AA expressed in baby hamster kidney cells revealed the absence of posttranslationally modified amino acid. Approximately 50% of the proteins were shortened by two to three amino acid residues at the C-terminus. Trypsin treatment of BHK rPDGF-AA lead to the identification of two internal epitopes that correspond to the two previously described domains in rPDGF-BB [Vogel, S., & Hoppe, J. (1989) Biochemistry 28, 2961-2966]. Cysteine residues at positions 37, 46, 47, and 93, respectively, were converted by site-directed mutagenesis into serine residues, and the monomeric proteins were prepared through expression in Escherichia coli. None of the mutant proteins was able to dimerize, but all of them exhibited to various extents a reversible conformational change which may reflect an intermediate prefolded monomer. An intramolecular disulfide bridge between Cys-10 and Cys-91/93 was identified in these monomers. From a mixture of the mutant proteins 37 and 46, an active dimer was reconstituted, suggesting an intermolecular cysteine bridge between these two residues.

Preparation of biologically active platelet-derived growth factor isoforms AA and AB. Preferential formation of AB heterodimers.

We have expressed the mature platelet-derived growth factor (PDGF) A chain within a fusion protein of the cro repressor and beta-galactosidase in Escherichia coli. Monomeric PDGF-A was excised from this fusion protein by CNBr cleavage. After protection of thiols by S-sulfonation, this fragment was purified by gel permeation chromatography and reversed-phase high-performance liquid chromatography. The monomeric protein was dimerized in the presence of a mixture of reduced and oxidized glutathione to yield biologically active recombinant AA dimer (rPDGF-AA) with an overall yield of about 0.2 mg/l culture. When monomeric rPDGF-A and rPDGF-B were reacted at stoichiometric concentrations in the presence of glutathione, almost exclusively hetero-dimers of type AB were formed. Heterodimers AB stimulated [3H]thymidine incorporation into AKR-2B fibroblasts half-maximally at about 2 ng/ml. AA homodimers were fivefold less active. About 60,000 binding sites were found for rPDGF-AB, 30,000 for rPDGF-AA and 45,000 for rPDGF-BB on AKR-2B fibroblasts.

Large-scale preparation of recombinant platelet-derived growth factor AA secreted from recombinant baby hamster kidney cells.

The short isoform of platelet-derived growth factor A (PDGF-A) was expressed in a mammalian host (BHK-21 cell). A cell line was obtained that secreted up to 0.3 micrograms/10(6) cells recombinant PDGF-A chain homodimer/day into the medium. For large-scale production of supernatant, cells were grown either in roller bottles or in 2.5-1 stirred tank fermenters. A simple two-step procedure was developed to purify recombinant PDGF-AA (rPDGF-AA). The first step was adsorption onto porous glass and the final step was reversed-phase high-performance liquid chromatography. The yield was 0.2 mg/l supernatant. A total amount of 20-30 mg pure rPDGF-AA may be obtained from a single fermenter run. Sequence analysis showed the correct amino terminus and no internal proteolytic cleavages. The specific activity was 5 ng/ml for mouse AKR-2B cells. [125I]rPDGF-AA had an affinity constant of approximately 0.5 nM to these cells and 25,000 binding sites were estimated/cell.

Preparation of biologically active platelet-derived growth factor type BB from a fusion protein expressed in Escherichia coli.

Preparations of the mitogen platelet-derived growth factor (PDGF) from human platelets contain two related polypeptides termed A chain and B chain. PDGF-B is highly homologous to a portion of p28v-sis, the transforming protein of simian sarcoma virus. We have studied the mitogenic potential of a PDGF-BB-like homodimer by expressing the sequence coding for the mature part of PDGF-B in Escherichia coli. Expression was achieved as cro-beta-gal-PDGF-B fusion protein which was exclusively found in the "inclusion bodies". A monomeric PDGF-B fragment shortened by 12 amino acid residues from the NH2 terminus was excised from the fusion protein by CNBr cleavage. After protection of thiols by S-sulfonation, this fragment was purified by gel permeation chromatography and reversed-phase high-performance liquid chromatography. This monomeric protein was dimerized in the presence of a mixture of reduced and oxidized glutathione to yield biologically active rPDGF-BB with an overall yield of approximately 0.7 mg of rPDGF-BB/L of culture. Escherichia coli rPDGF-BB stimulated [3H]thymidine incorporation into AKR2B fibroblast at concentrations of about 1 ng/mL.

The long 3'-untranslated regions of the PDGF-A and -B mRNAs are only distantly related.

A cDNA clone of about 2300 base pairs was prepared from the human osteosarcoma cell line U-2 OS by hybridization with a 22-mer oligonucleotide complementary to the NH2-terminus of PDGF-A. Restriction and sequence analysis showed that this clone contains the entire coding region for PDGF-A and a long 3'-untranslated region which is only distantly related to that in the mRNA of PDGF-B.

Renal oncocytoma. A cytogenetic and morphologic study.

A detailed G-banded chromosome analysis was performed in primary cell cultures obtained from two renal oncocytomas. Both tumors were characterised by clonal chromosome aberrations. One had a stemline karyotype 45,X,-X,rcp(8;19),der(13)t(X;13) and several sidelines with variant additional aberrations. The chromosome analysis of the other neoplasm revealed a pseudodiploid stemline 46,XY,del(1q),der(13)t(1;13) and cells with normal karyotype; both types of cells showed several telomeric associations of chromosomes, formed mostly by smaller chromosomes. The chromosome pattern of oncocytomas is discussed in comparison with the cytogenetics of renal cell carcinomas.