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G Goldberger

Publications and source records attributed to G Goldberger.

At least 19 recordsLinked to original sources
PubMed1999

Cloning and characterization of the non-catalytic heavy chain of mouse complement factor I gene: structure comparison with the human homologue.

The gene sequence encoding the non-catalytic heavy chain of mouse complement factor I (mCFI) was cloned and its exon-intron organization and domain structure characterized. The genomic organization of mCFI differs in several aspects from its human homologue (hCFI). The intron sizes are remarkably different. Exons 2 and 4 in mCFI are larger than their counterparts in hCFI by 9 bp and 6 bp respectively. Whereas the diversity (D) region of hCFI is encoded by two exons (exon 7 or hD2 and exon 8 or hD4), this region in mCFI is encoded by three exons; exon 6A or mD1 (located at the 3'-end of the LDLr A2 domain), exon 7 or mD2 and exon 8, an extended exon (56 bp) composed of mD3, fused upstream of mD4. In contrast, hCFI lacks D1 and D3 subregions and exon 8 in hCFI consists of only hD4, 36 bp in length. Thus the heavy chain of mCFI is organized into 10 exons compared to 9 exons in hCFI.

Amino Acid Sequence↗
PubMed1998

Cloning and characterization of the promoter for the human complement factor I (C3b/C4b inactivator) gene.

Complement factor I is a serine proteinase that regulates the classical and alternative pathways of complement by cleaving C3b and C4b and preventing the assembly of C3 and C5 convertase enzymes. In order to understand the regulation of factor I gene expression in liver cells, 4kb of the 5' flanking region of the gene was cloned, and the 1474-bp 3'-end was sequenced and shown to contain a number of transcription factor consensus sequences. A major and two minor transcription start sites were identified, respectively, at 152, 178, and 198bp upstream of the translation start site by primer extension analysis. The transcriptional activity of the 1474-bp fragment was analyzed by fusion of 5' deletion constructs to a cat-encoding gene expression vector and transient transfections into Hep G2 cells. A 273-bp fragment located at -112 to +161 relative to the major transcription start site was sufficient for promoter activity. The 3' fragment spanning +3 to +161 and containing a TATA-like element did not demonstrate promoter activity, suggesting that the core promoter resides in a 115-bp sequence located between -112 and +3. This region contains an Inr-like element overlapping the major cap site and a CTF-NF1 element, two potential CCAAT boxes and an AP-2 element partially overlapping an Sp-1 site. Thus, factor I promoter may belong to the TATA-less Inr-driven class II promoters whose transcription is regulated by Sp-1. The transcriptional activity of the 1474-bp 5' flanking fragment was upregulated by PMA, IL-6 and TNF-alpha, suggesting that factor I may be an acute phase reactant.

Base Sequence↗
PubMed1996

cDNA cloning, sequencing and chromosomal assignment of the gene for mouse complement factor I (C3b/C4b inactivator): identification of a species specific divergent segment in factor I.

Factor I is an essential regulatory serine proteinase of the complement cascade. It cleaves and inactivates the C3b and C4b constituents of the C3 and C5 convertases and thereby regulates many complement-mediated activities. The human protein is a heterodimer composed of a 50 kDa non-catalytic subunit (which contains several domains, i.e. FIM, CD5, LDLr type A) disulfide linked to a 38 kDa catalytic subunit. Recent characterization of Xenopus factor I cDNA revealed a 29 residue negatively charged region in its heavy chain which is absent in the human protein (Kunnath-Muglia et al., Molec. Immun. 30, 1249-1256, 1993). We report the complete cDNA sequence of mouse factor I as well as a partial chicken factor I cDNA sequence. Alignment of these two sequences with the published sequences for human and Xenopus proteins (a) demonstrates an overall conservation of primary structure and domain organization of mouse factor I, and (b) defines a divergent segment (D segment) in each species. In Xenopus protein, the D segment includes the 29 residue negatively charged region. In each of the four species examined, the D segment differed in length, sequence, organization, and number of repeated subregions. These differences reflect a considerable evolution of D segment. The significance of the diversity of the D segment is at present unclear. We also report the chromosomal localization of the mouse factor I gene (Cfi) to distal chromosome 3 near Egf.

Amino Acid Sequence↗
PubMed1995

Processing of human factor I in COS-1 cells co-transfected with factor I and paired basic amino acid cleaving enzyme (PACE) cDNA.

Factor I is an active serine proteinase in plasma that regulates both the classical and alternative complement pathways by cleaving C3b and C4b thereby preventing the assembly of C3 and C5 convertase enzymes. In this study, a full-length human factor I cDNA was cloned into the pMT2 expression vector and the pMT2-fI construct was expressed transiently in COS-1 cells and stably in CHO-K1 cells. The transfected COS-1 cells secreted large amounts of recombinant pro-factor I (85 kD). Co-transfection of COS-1 cells with pMT2-fI and the cDNA expression plasmid for PACE (paired basic amino acid cleaving enzyme), resulted predominantly in the secretion of a proteolytically processed form of recombinant factor I (heavy chain, 47 kD; light chain, 35 kD). Following co-transfection of pMT2-fI and pSVNeo.1 into CHO-K1 cells and selection in medium containing G418, a stably transfected clone was isolated that secreted pro-factor I (85 kd) and proteolytically processed factor I (heavy chain, 48 kD; light chain, 37 kD) in approximately equal amounts. The molecular sizes of the subunit chains of the expressed factor I were generally slightly smaller than those of human plasma factor I. The activity of recombinant factor I present in the culture supernatants of transfected COS-1 and CHO-K1 cells was assayed by its ability to cleave 125I-C3b in the presence of factor H and was found to be low when compared with factor I purified from human plasma. However, since the functional activity of purified factor I was reduced approximately 50% in the presence of conditioned medium from non-transfected cells, it is suggested that the cold C3b present in the factor I-deficient serum used to supplement the culture medium probably competed with the 125I-C3b tracer, thereby decreasing the sensitivity of the assay for the recombinant factor I proteins.

Animals↗
PubMed1991

Biosynthesis and secretion of rat salivary proteins by Xenopus laevis oocytes.

Xenopus laevis oocytes injected with poly (A+) RNA isolated from rat parotid and submandibular glands synthesize and secrete salivary proteins. Amylase was identified in the media of cultured oocytes injected with rat parotid mRNA by size and immunoprecipitation with anti-human amylase serum. Secretion of the salivary proteins was detectable in the medium eight h following the parotid mRNA injection and continued in a time-dependent fashion for up to 96 h. In contrast to rat parotid slices in culture, which demonstrate a regulated pathway of secretion highly responsive to the secretagogue isoproterenol, secretion of salivary proteins by oocytes did not respond to the stimulation by isoproterenol. Though parotid mRNA is presumed to contain the templates encoding the regulated pathway of secretion, reconstitution of this pathway of secretion in oocytes was not observed in our experiments. Since Xenopus laevis oocytes secrete constitutively significant amounts of proteins when injected with salivary gland mRNA, they are a useful biological system for the analysis of secretion, processing, and function of salivary proteins.

Animals↗
PubMed1989

Mapping of the human complement factor I gene to 4q25.

A detailed genetic and physical map of human complement factor I (IF) using somatic cell hybrids, in situ hybridization, and genetic linkage is reported. The gene has been localized to band 4q25. The order GC-INP10-ADH3-EGF-IF-IL2-MNS is proposed for genes on 4q on the basis of genetic and physical mapping techniques. A BclI polymorphism found with the IF probe demonstrated a maternal origin for a de novo deletion of chromosome 4 that was used in physically mapping the gene. The genetic and physical distances around band 4q24 suggest that 1 cM is approximately 1.2 million bp of DNA. This work provides a useful addition to the map of 4q.

Chromosome Mapping↗
PubMed1987

Effect of hydroxyorganoboranes on synthesis, transport and N-linked glycosylation of plasma proteins.

Utilizing a recently developed method (Boradeption) for transferring water-insoluble hydroxyorganoborane compounds into the cells, we observed inhibition of protein synthesis by three of these compounds and inhibition of secretion of plasma proteins by four of them in human hepatoma HepG2 cells. These effects were specific in that the cell viability was not affected and an increase in protein catabolism was not observed. Three compounds caused a compound-specific alterations in the electrophoretic mobility of secreted glycoproteins due to underlying changes in the N-linked carbohydrate moieties. Results presented suggest a potential new source of cellular probes.

Blood Proteins↗
PubMed1987

Human complement factor I: analysis of cDNA-derived primary structure and assignment of its gene to chromosome 4.

Factor I is a serine proteinase of complement which together with one of several specific cofactors cleaves activation products of the third and fourth components of complement (C3b and C4b) and modulates the activity of C3 convertase. A heterodimer glycoprotein (Mr = 88,000), factor I is synthesized as a single-chain precursor, prepro-I, which undergoes intracellular proteolytic processing. The human hepatoma line HepG2, however, secretes predominantly the single-chain precursor pro-I. In order to determine the molecular basis for this apparent processing defect, factor I cDNA clones were isolated from a HepG2 mRNA-derived library. Sequencing of the largest insert, HI1971, revealed that it contains 14 base pairs of 5' untranslated region, the complete coding sequence for the 583-residue prepro-I (NH2-signal peptide-heavy chain-linking peptide-light chain-COOH), two polyadenylation signals within the 200-base pair 3' untranslated region, and a portion of poly(A) tail. Analysis of the derived protein structure 1) reveals a mosaic multidomain structure of the heavy chain; 2) demonstrates structural similarity between intracellular conversion of pro-I and activation of other serine proteinase zymogens; and 3) indicates that the light chain of factor I resembles most closely the active subunit of tissue plasminogen activator among all serine proteinases and factor D among complement proteinases. Furthermore, this protein sequence was compared to the sequences of factor I cDNA clones isolated from normal human liver libraries and found to be identical. By exclusion, this defines as cellular the basis for the inefficient processing of pro-I by the HepG2 line. Chromosomal localization by the somatic cell hybrid method maps the factor I gene to chromosome 4.

Amino Acid Sequence↗
PubMed1987

Transcriptional regulation of genes encoding the acute-phase proteins CRP, SAA, and C3.

Inflammation or acute tissue injury results in a programmed change in the concentration of several plasma proteins. Among these proteins, two--C-reactive protein (CRP) and serum amyloid A protein (SAA)--increase up to 1000-fold after an acute-phase stimulus in humans and rabbits. To determine the mechanism for regulation of acute-phase gene expression, we examined changes in the rates of transcription and specific hepatic mRNA content for rabbit CRP, SAA, and some complement protein mRNA during an acute-phase response. Induction of a sterile inflammatory reaction with intramuscular injection of turpentine resulted in an increase in the hepatocellular content of CRP, SAA, C3, and factor B mRNA and the transcription of CRP, SAA, and C3 genes. These data suggest that the increase in CRP, SAA, and C3 serum concentrations observed during an acute-phase reaction is due to an increase in biosynthesis and is, at least in part, under transcriptional control.

Acute-Phase Proteins↗
PubMed1986

Regulation of class III major histocompatibility complex gene products by interleukin-1.

Interleukin-1 (IL-1) is a product of mononuclear phagocytes that mediates changes characteristic of the response to inflammation or tissue injury (the acute-phase response). One of two structurally and functionally homologous major histocompatibility complex (MHC) class III genes encodes a positive acute-phase protein, complement factor B. The closely linked complement C2 gene is not affected during the acute-phase response. Purified human IL-1, pH 7.0, and recombinant-generated murine IL-1, pH 5.0, increased the expression of factor B and other positive acute-phase proteins in human hepatoma cells but decreased the expression of albumin, a negative acute-phase reactant. Furthermore, in a murine fibroblast L-cell line transfected with cosmid DNA bearing the human C2 and factor B genes, IL-1 mediated a reversible dose- and time-dependent increase in factor B expression in the transfected cells. Expression of the C2 gene was not affected by IL-1. The effect of IL-1 on factor B expression involves a mechanism acting at a pre-translational level as demonstrated by an increase in specific messenger RNA content and a corresponding increase in biosynthesis and secretion of factor B. The structural basis and mechanism for selective and independent regulation of these genes provides insight into the molecular control of the inflammatory response.

Animals↗
PubMed1985

Human serum amyloid A (SAA): biosynthesis and postsynthetic processing of preSAA and structural variants defined by complementary DNA.

To study structural variants of human serum amyloid A (SAA), an apoprotein of high-density lipoprotein, complementary DNA clones were isolated from a human liver library with the use of two synthetic oligonucleotide mixtures containing sequences that could code for residues 33-38 and 90-95 of the protein sequence. The SAA-specific cDNA clone (pA1) contains the nucleotide sequence coding for the mature SAA and 10 amino acids of the 18-residue signal peptide. It also includes a 70 nucleotide long 3'-untranslated region and approximately 120 bases of the poly(A) tail. The derived amino acid sequence of pA1 is identical with the alpha form of apoSAA1. A fragment of pA1 containing the conserved (residues 33-38) region of SAA also hybridized with RNA from human acute phase liver and acute phase stimulated, but not unstimulated, mouse and rabbit liver. In contrast, a fragment corresponding to the variable region hybridized to a much greater extent with human than with rabbit or murine RNA. Human acute phase liver SAA mRNA (approximately 600 nucleotides in length) directs synthesis of preSAA (Mr 14 000) in a cell-free translating system. In a Xenopus oocyte translation system preSAA is synthesized and processed to the mature Mr 12 000 product. The complete 18 amino acid signal peptide sequence of preSAA was derived from sequencing cDNA synthesized by "primer extension" from the region of SAA mRNA corresponding to the amino terminus of the mature product. Two other SAA-specific cDNA clones (pA6 and pA10) differed from pA1 in that they lack the internal PstI restriction enzyme site spanning residues 54-56 of pA1.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1985

Tissue-specific pretranslational regulation of complement production in human mononuclear phagocytes.

The net production of the complement protein C2, C4, and factor B differ among mononuclear phagocytes from peripheral blood and different tissues in experimental animals and in humans. To examine the mechanisms that regulate these differences in humans, the proportions of C2-producing cells, the average single-cell production rate of C2, the posttranslational glycosylation and kinetics of secretion of C2 and factor B, and the amounts of C2 and factor B mRNA were examined in freshly isolated peripheral blood monocytes, monocytes maintained up to 2 wk in culture, and freshly isolated tissue macrophages from breast milk and bronchoalveolar lavage. In addition, the biosynthesis of two other proteins synthesized and secreted by mononuclear phagocytes, C3 and lysozyme, were examined. We report that despite comparable rates of C3 and lysozyme synthesis and similar processing and kinetics of secretion of C2 and factor B, the freshly isolated tissue macrophage differs from the monocyte-derived macrophage in the proportion of C2-producing cells, in the average single-cell production rate of complement, and in the amounts of specific C2 and factor B mRNA. These differences are tissue specific, because C2-specific mRNA content in bronchoalveolar macrophages is considerably greater than in breast milk macrophages, although the amounts of factor B mRNA are comparable. These data suggest that tissue-specific regulation of complement production in human mononuclear phagocytes occurs at a pretranslational level. These studies now provide a basis for investigation of the molecular effects of agents that modulate the biologic functions of monocytes and macrophages.

Cell Differentiation↗
PubMed1984

Distinct primary translation products from human liver mRNA give rise to secreted and cell-associated forms of complement protein C2.

The second component of complement (C2), is a class III major histocompatibility complex gene product and a glycoprotein in the classical complement activating system. Synthesis in the human hepatoma-derived cell line HepG2 results in three intracellular forms: an 84-kDa form secreted in 1-2 h; 79-kDa and 70-kDa forms that remain cell-associated for intervals up to 12 h. All three forms are C2 polypeptides as demonstrated by inhibition of immunoprecipitation with unlabeled C2 and the presence of common major peptide fragments following chymotryptic digestion. The cell-associated forms of C2 are not products of proteolysis as demonstrated by experiments with multiple proteinase inhibitors and by observations of the kinetics of synthesis. Inhibition of core glycosylation by tunicamycin and deglycosylation by acid hydrolysis indicate that the three intracellular C2 polypeptides are glycosylated to a similar extent. Although the 84-kDa form of C2 is susceptible to C1s cleavage, the two cell-associated forms are not. Cell-free biosynthesis by mRNA from HepG2 or human liver results in three primary translation products corresponding to the three unglycosylated forms of C2. These results indicate that HepG2 cells synthesize C2 protein in both secreted and cell-associated forms and that each form is derived from a separate primary translation product.

Carcinoma, Hepatocellular↗
PubMed1984

Biosynthesis and postsynthetic processing of human C3b/C4b inactivator (factor I) in three hepatoma cell lines.

Human factor I is a two-chain plasma glycoprotein composed of disulfide-linked 50,000- and 38,000-dalton subunits. Analysis of its biosynthesis and postsynthetic processing demonstrated that factor I is synthesized as a single chain precursor (pro-I) that undergoes glycosylation and limited proteolysis to generate the native protein. One of three human hepatoma cell lines, HepG2 , secreted factor I predominantly (70-90%) in a single chain pro-I form. The other cell lines secrete factor I predominantly in its two chain native form. The defect in conversion of pro-I to I in HepG2 was protein specific since other multichain proteins, derived from single chain precursors, the third, fourth, and fifth components of complement were processed normally. Further analysis of the inefficient pro-I to I conversion by HepG2 revealed that Xenopus oocytes injected with HepG2 mRNA secreted factor I in a predominantly two-chain form. In addition, the apparent sizes of native factor I, transferrin, and alpha-1-antitrypsin secreted by the three hepatoma lines differed due to differences in postsynthetic processing.

Animals↗
PubMed1984

Synthesis, intracellular processing, and signal peptide of human apolipoprotein E.

Northern blotting analysis has shown apo-E mRNA synthesis by human liver, HepG2 cells, and primary cultures of human monocyte macrophages but not by the macrophage-like cell line U937 and normal or transformed human fibroblasts. Cell-free translation has shown that the primary translation product of apo-E consists of one major and one minor isoprotein of apparent Mr = 28,500 and isoelectric points 6.20 and 6.02, respectively. These isoproteins differ by +1 and 0 charges from apo-E3 and have been designated preapo-E. Co-translational treatment of mRNA with dog pancreatic membranes converts both preapo-E isoproteins to a form which is undistinguishable by two-dimensional gel electrophoresis from plasma apo-E3. The isolation and nucleotide sequence analysis of a full length apo-E cDNA clone has shown that preapo-E contains an 18-amino acid NH2-terminal signal peptide compared to plasma apo-E. The signal peptide sequence is: MetLysValLeuTrpAlaAlaLeuLeuValThrPheLeuAlaGlyCysGlnAla. Comparison of co-translationally modified apo-E with intracellular, secreted, and plasma forms indicates that after the intracellular cleavage of the signal peptide, the protein is glycosylated with carbohydrate chains containing sialic acid, secreted as sialoapo-E (apo-Es), and subsequently desialated in plasma. These findings demonstrate that apo-E is synthesized as preprotein and undergoes intracellular proteolysis and glycosylation and extracellular desialation to attain the major asialoapo-E isoprotein form observed in plasma.

Amino Acid Sequence↗
PubMed1983

Use of a cDNA clone for the fourth component of human complement (C4) for analysis of a genetic deficiency of C4 in guinea pig.

A cDNA clone for the fourth complement component (C4), pC4AL1, has been isolated from a human adult liver cDNA library by using a synthetic oligonucleotide mixture containing all 384 possible sequences coding for residues 14-21 of the C4 gamma-chain amino acid sequence. This clone spans the entire C4 gamma-chain coding sequence and includes a short 3' untranslated region, a poly(A) recognition site, and 16 nucleotides of the poly(A) tail. The 5' end of the clone begins 18 nucleotides upstream from the amino terminus of the C4 gamma chain and codes for Arg-Asn-Arg-Arg-Arg-Arg, a highly charged proteolytic cleavage site involved in the processing of pro-C4 to native C4. Liver mRNA preparations from C4-deficient guinea pigs were incapable of directing synthesis of pro-C4 or C4 peptides in cell-free translation experiments. Southern blot analysis using pC4AL1 as a hybridization probe of C4-deficient guinea pig DNA established that the deficiency is not the result of deletion of the entire C4 gene. RNA blot analysis using pC4AL1 as a hybridization probe of normal guinea pig liver mRNA revealed a C4 mRNA of 5.0 kilobases (kb). No such mRNA species was observed in C4-deficient guinea pig liver mRNA; however, a 7.0-kb RNA was detected, indicating the presence of a C4 precursor RNA. These results suggest that the basis of C4 deficiency in the guinea pig is a post-transcriptional defect in the processing of C4 precursor RNA to mature C4 mRNA.

Amino Acid Sequence↗
PubMed1983

Molecular map of the murine S region.

Eighteen overlapping cosmid clones spanning 240 kilobases and encoding the gene for factor B and two genes related to the fourth component of complement (C4) were isolated from a murine H-2d genomic library. Cosmid clones were identified by hybridization to human cDNA probes for factor B and C4 and were linked by chromosomal walking procedures. The cluster of clones contains two regions with sequences homologous to the C4 cDNA probe, both in the same orientation, representing a direct duplication of at least 55 kilobases of chromosomal DNA, separated by a shorter (less than 25 kilobases) segment of nonduplicated DNA. Restriction fragment-length polymorphism seen by using C4 probes maps these sequences to the S region of the major histocompatibility complex. 5' to the two C4-like sequences is an approximately equal to 40-kilobase-long region of chromosomal DNA remarkable for its lack of restriction fragment-length polymorphism, containing sequences homologous to the human factor B cDNA probe. These experiments demonstrate that the structural gene for factor B is located in the S region of the murine major histocompatibility complex and that this region contains an extensive direct duplication that contains the structural gene for mouse C4 and, we presume, for the sex-limited protein variant, Slp. RNA transfer blot analysis of total liver RNA from high C4- and low C4-producing strains showed that steady-state levels of C4-hybridizing RNA were much greater in high C4-producing strains. Regulation of circulating C4 levels in high C4 and low C4 strains is at least partly at the level of mRNA transcription, processing, or degradation.

Animals↗