Biomedical subjects

M L Koschinsky

Publications and source records attributed to M L Koschinsky.

49 records · Page 3Linked to original sources

PubMed1994

Expression and characterization of apolipoprotein(a) kringle IV types 1, 2 and 10 in mammalian cells.

We have designed expression constructs containing sequences corresponding to apolipoprotein(a) kringle IV types 1, 2 and 10 and used these constructs to transfect human embryonic kidney cells. We have also expressed a mutant form of kringle IV type 2 in which the N-linked glycosylation site has been removed by replacement of an asparagine residue with an alanine. Immunoprecipitation analysis of [35S]Cys-labeled transfected cell culture supernatants resulted in the observation of two bands for kringle IV type 1 (M(r) approximately 30,000 and 26,000), two bands for kringle IV type 2 (M(r) approximately 25,000 and 22,000), two bands for kringle IV type 10 (M(r) approximately 27,000 and 23,000) and one band for the glycosylation mutant (M(r) approximately 22,000). In all cases, observed molecular weights greatly exceeded those predicted from amino acid sequence, suggesting the presence of both N- and O-linked glycans. None of the recombinant single kringles were observed to bind to fibrinogen as determined by ELISA or by co-immunoprecipitation in the case of kringle IV type 10 and only kringle IV type 10 was able to bind to lysine--Sepharose. These data suggest that apo(a) binding to fibrinogen/fibrin may require motif(s) in addition to apo(a) kringle IV type 10.

Amino Acid Sequence↗

PubMed1994

Analysis of structure--function relationships in human apolipoprotein(a).

Elevated levels of lipoprotein(a) (Lp(a)) have been strongly correlated with the development of atherosclerosis in human populations. Lp(a) is distinguishable from low density lipoprotein by the presence of the unique protein component apolipoprotein(a) (apo(a)), which contains repeated domains that closely resemble that of plasminogen kringle IV. Using human embryonic kidney cells, we have expressed a recombinant form of apo(a) (r-apo(a)) containing 17 kringle IV-like domains. We have utilized this recombinant expression system to study the assembly of Lp(a) particles. We have demonstrated that Lp(a) particles containing r-apo(a) can be assembled extracellularly in plasma by covalent linkage to low density lipoprotein. Using site-directed mutagenesis, we have demonstrated that a cysteine residue present at position 4057 of the apo(a) protein (i.e., in the penultimate kringle IV repeat) mediates this covalent linkage. Using polymerase chain reaction amplification of liver apo(a) complementary DNA, we have demonstrated the presence of a polymorphism in apo(a) kringle IV type 10, which results in the substitution of a threonine for a methionine. Preliminary studies indicate that the presence of a threonine at this position may enhance the interaction of Lp(a) with lysine-Sepharose.

Animals↗

PubMed1994

Binding of recombinant apolipoprotein(a) to extracellular matrix proteins.

Elevated levels of lipoprotein(a), which consists of apolipoprotein(a) [apo(a)] covalently linked to a low-density lipoprotein-like moiety, is an independent risk factor for the development of atherosclerosis. We show that a recombinant form of apo(a) [r-apo(a)] binds strongly to fibronectin and fibrinogen, weakly to laminin, and not at all to von Willebrand factor, vitronectin, or collagen type IV. In contrast to the binding of plasminogen to fibronectin, r-apo(a) binding does not appear to be mediated by lysine-dependent interactions, based on the inability of epsilon-aminocaproic acid concentrations up to 0.2 mol/L to significantly decrease r-apo(a) binding to fibronectin. Plasminogen competed weakly for the binding of r-apo(a) to fibronectin, whereas r-apo(a) completely abolished plasminogen binding. The 29- and 38-kd heparin-binding thermolysin fragments of fibronectin, previously identified as the lipoprotein(a) binding domains, were digested with trypsin, and a peptide that retained the ability to bind r-apo(a) was isolated; the sequence of the peptide (AVTTIPAPTDLK) corresponds to the amino terminus of the 29- and 38-kd domains. A synthetic peptide with this sequence was able to compete effectively with fibronectin for r-apo(a) binding.

Aminocaproates↗

PubMed1993

Identification of the cysteine residue in apolipoprotein(a) that mediates extracellular coupling with apolipoprotein B-100.

We have utilized a recombinant expression system in order to study the assembly of lipoprotein(a) (Lp(a)) particles. Using a 17-kringle recombinant form of apolipoprotein(a) (apo(a)) to transiently transfect human hepatoma cells, we could not detect recombinant Lp(a) (r-Lp(a)) particles intracellularly, by analysis of postnuclear lysates. However, covalent r-Lp(a) complexes were observed in the transfected cell supernatants. Upon addition of [35S]Cys-labeled human embryonic kidney cell supernatants transfected with 9-kringle or 17-kringle recombinant apo(a) (r-apo(a)) variants to human plasma, covalent r-Lp(a) complexes were observed, which could be immunoprecipitated using antibodies specific for either apo(a) or apolipoprotein B-100 (apoB-100); r-Lp(a) complexes containing the 17-kringle r-apo(a) were shown to be in the 1.063 g/ml < d < 1.20 g/ml range by density gradient ultracentrifugation analysis. Complexes containing the 17-kringle r-apo(a) formed rapidly within 20 min, with a slow increase observed up to 90 min. Addition of increasing amounts of plasma, as well as increasing amounts of isolated human low density lipoprotein to cell culture supernatants containing [35S]Cys-labeled 17-kringle r-apo(a) led to enhanced r-Lp(a) complex formation. Blocking of free sulfhydryls in apo(a) with N-ethylmaleimide resulted in inhibition of r-Lp(a) complex formation in plasma, verifying the role of free sulfhydryls in Lp(a) particle assembly. Using site-directed mutagenesis, we demonstrated that Cys4057 in apo(a) is involved in disulfide linkage with apoB-100 in Lp(a) particles.

Amino Acid Sequence↗

PubMed1993

The apolipoprotein(a) kringle IV repeats which differ from the major repeat kringle are present in variably-sized isoforms.

Elevated levels of plasma lipoprotein(a) [Lp(a)] have been correlated with the development of atherosclerosis in human populations. Apolipoprotein(a) [apo(a); the distinguishing protein component of Lp(a)] is characterized by multiple repeats of a sequence that closely resembles kringle IV of plasminogen. Variably-sized Lp(a) isoforms that are observed in the human population have been shown to occur as a result of differences in the numbers of the repeated kringle IV units in apo(a). Using PCR analysis of human liver mRNA, we have analyzed apo(a) from 10 unrelated individuals in order to determine the presence or absence of kringle IV repeat #1, and #30-#37. Based on the apo(a) cDNA sequence published for one individual, these kringles all differ to some degree in amino acid sequence from the major kringle IV repeat, which is present in a number of identically repeated copies. We found that sequences corresponding to apo(a) kringle IV repeat #1, and #30-#37 were present in all individuals studied. This suggests that the inverse relationship that has been observed between Lp(a) isoform size and plasma Lp(a) levels is mediated by different numbers of identical kringle IV repeats, by an as yet undetermined mechanism. During the course of this study, we identified a Met-->Thr polymorphism in the apo(a) kringle IV repeat #37. The calculated frequencies of the Met and Thr alleles were 0.58 and 0.42 respectively. We did not observe a correlation between the Met-->Thr substitution and either plasma Lp(a) levels, or apo(a) transcript size.

Amino Acid Sequence↗

PubMed1992

Reconstitution of lipoprotein(a) by infusion of human low density lipoprotein into transgenic mice expressing human apolipoprotein(a).

Lipoprotein(a) (Lp(a)) is an atherosclerosis-causing lipoprotein that circulates in human plasma as a complex of low density lipoprotein (LDL) and apolipoprotein(a) (apo(a)). It is not known whether apo(a) attaches to LDL within hepatocytes prior to secretion or in plasma subsequent to secretion. Here we describe the development of a line of mice expressing the human apo(a) transgene under the control of the murine transferrin promoter. The apo(a) was secreted into the plasma, but circulated free of lipoproteins. When human (h)-LDL was injected intravenously, the circulating apo(a) rapidly associated with the lipoproteins, as determined by nondenaturing gel electrophoresis. Human HDL and mouse LDL had no such effect. When h-VLDL was injected, there was a delayed association of apo(a) with the lipoprotein fraction which suggests that apo(a) preferentially associated with a metabolic product of VLDL. The complex of apo(a) with LDL formed both in vivo and in vitro was resistant to boiling in the presence of detergents and denaturants, but was resolved upon disulfide reduction. These studies suggest that apo(a) fails to associate with mouse lipoproteins due to structural differences between human and mouse LDL, and that Lp(a) formation can occur in plasma through the association of apo(a) with circulating LDL.

Aminocaproates↗

PubMed1992

Apolipoprotein(a) and plasminogen interactions with fibrin: a study with recombinant apolipoprotein(a) and isolated plasminogen fragments.

Lipoprotein(a) [Lp(a)], but not low-density lipoprotein (LDL), was previously shown to impair the generation of fibrin-bound plasmin [Rouy et al. (1991) Arterioscler. Thromb. 11, 629-638] by a mechanism involving binding of Lp(a) to fibrin. It was therefore suggested that the binding was mediated by apolipoprotein(a) [apo(a)], a glycoprotein absent from LDL which has a high degree of homology with plasminogen, the precursor of the fibrinolytic enzyme plasmin. Here we have evaluated this hypothesis by performing comparative fibrin binding studies using a recombinant form of apo(a) containing 17 copies of the apo(a) domain resembling kringle 4 of plasminogen, native Lp(a), and Glu-plasminogen (Glu1-Asn791). Attempts were also made to identify the kringle domains involved in such interactions using isolated elastase-derived plasminogen fragments. The binding experiments were performed using a well-characterized model of an intact and of a plasmin-digested fibrin surface as described by Fleury and Anglés-Cano [(1991) Biochemistry 30, 7630-7638]. Binding of r-apo(a) to the fibrin surfaces was of high affinity (Kd = 26 +/- 8.4 nM for intact fibrin and 7.7 +/- 4.6 nM for plasmin-degraded fibrin) and obeyed the Langmuir equation for adsorption at interfaces. The binding to both surfaces was inhibited by the lysine analogue AMCHA and was completely abolished upon treatment of the degraded surface with carboxypeptidase B, indicating that r-apo(a) binds to both the intrachain lysines of intact fibrin and the carboxy-terminal lysines of degraded fibrin. As expected from these results, both r-apo(a) and native Lp(a) inhibited the binding of Glu-plasminogen to the fibrin surfaces.(ABSTRACT TRUNCATED AT 250 WORDS)

Apolipoproteins↗

PubMed1991

Apolipoprotein(a): expression and characterization of a recombinant form of the protein in mammalian cells.

We have stably expressed a recombinant form of apo(a) in a human embryonic kidney cell line. The engineered protein (predicted mass of 250 kDa) contains 17 copies of the apo(a) domain, which resembles kringle 4 of plasminogen, followed by the plasminogen-like kringle 5 and protease-like domain of apo(a). The recombinant protein [r-apo(a)] was isolated from cell culture media by immunoaffinity chromatography, and its physical properties were studied. As is the case for apo(a) isolated from plasma-derived Lp(a), r-apo(a) is highly glycosylated (23% by weight), containing both N- and O-linked glycans, which results in an observed molecular mass of 500 kDa by SDS-PAGE. The high sialic acid content was reflected in a pI of 4.3 for the r-apo(a). Two subpopulations of r-apo(a) secreted by the permanent cell line were identified with respect to lysine-Sepharose binding; the majority of the r-apo(a) bound specifically to this matrix and was eluted with epsilon-aminocaproic acid (epsilon-ACA). When the r-apo(a) plasmid was used to transfect a human hepatoma cell line, lipoprotein particles were secreted containing the disulfide-linked complex of apoB-100 and the r-apo(a). The density of these particles was shown to be heterogeneous, with the majority of the r-Lp(a) floating in the density range of plasma-derived Lp(a).

Apolipoproteins↗

PubMed1990

Apolipoprotein(a) size heterogeneity is related to variable number of repeat sequences in its mRNA.

Plasma apolipoprotein(a) [apo(a)] shows considerable size heterogeneity, existing as discrete glycoprotein isoform variants that range in apparent molecular mass from approximately 400 to 800 kDa. To study the molecular basis of protein size variability, we have isolated liver RNA from individuals with different apo(a) isoforms, and identified apo(a)-specific transcripts using Northern blot analysis. Transcript sizes were shown to be variable (8.0-12 kb) and in all cases were closely correlated with protein masses (590-850 kDa) as determined from immunoblots. Thus, it is almost certain that apo(a) isoform size variation is due to allelic differences in the number of its tandemly repeated sequences of 114 amino acids that resemble kringle four of plasminogen. The high carbohydrate content of apo(a) makes true molecular weight estimations in SDS-PAGE gels difficult. However, a recombinant form of apo(a) containing 17 kringle repeats (calculated molecular mass of 250 kDa) migrates on SDS-PAGE gels only slightly below apoB-100, with an apparent molecular mass of approximately 500 kDa. Since smaller protein isoforms have been observed in the population, this suggests that plasma apo(a) isoforms contain from less than 17 to greater than 30 tandemly repeated kringle units.

Apolipoproteins A↗

PubMed1987

Isolation and characterization of a processed gene for human ceruloplasmin.

A processed pseudogene for human ceruloplasmin has been isolated that contains DNA corresponding to the functional gene sequence encoding the carboxy-terminal 563 amino acid residues and the 3' untranslated region. The pseudogene appears to have arisen from a processed RNA species, since intervening sequences coincident with those of the functional gene have been removed, with the exception of a short segment of intronic sequence which denotes the 5' boundary of the pseudogene. The nucleotide sequence of the pseudogene is highly homologous (97% sequence identity) with that of the wild-type gene, suggesting that pseudogene formation was a relatively recent evolutionary event. In addition to single base substitutions, there is a large 213 base pair (bp) deletion in the pseudogene sequence which corresponds to the location of an intron-exon junction in the functional gene. A 4 bp duplication that occurs at amino acid residue 683 of the wild-type coding sequence results in a frameshift mutation and introduces a premature translational termination codon at this point. This is concordant with the inability to detect a human liver transcript corresponding to the pseudogene by nuclease S1 mapping analysis. The 3' end of the pseudogene is characterized by a 62 bp segment composed mainly of repeated TC dinucleotides. On the basis of genomic Southern blot analysis performed under high-stringency conditions, the pseudogene that we have identified seems to comprise the only sequence in the human genome that is closely related to the wild-type gene. Using somatic cell hybridization, we have mapped the pseudogene to human chromosome 8.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗

PubMed1987

Human genes encoding prothrombin and ceruloplasmin map to 11p11-q12 and 3q21-24, respectively.

The gene for human prothrombin, or factor II (F2) has been assigned to 11p11-q12 by the combined use of a panel of somatic cell hybrid DNAs and in situ hybridization, using both cDNA and genomic probes. In addition, the cDNA probe for F2 recognizes a homologous sequence which has been tentatively mapped to the X chromosome. Similar approaches have been used to confirm the assignment of the ceruloplasmin gene, but to regionally localize it more proximally than previously reported (3q21-q24). These results provide further evidence that genes encoding the coagulation factors and related proteins are dispersed throughout the human genome.

Ceruloplasmin↗

PubMed1987

Molecular studies of ceruloplasmin deficiency in Wilson's disease.

Deficiency of serum ceruloplasmin is a characteristic biochemical abnormality of Wilson's disease, although the mechanism of this finding is unknown. Ceruloplasmin messenger RNA (mRNA) levels were therefore examined in five patients with Wilson's disease and five controls with other types of hepatic disease. Northern and dot blot hybridizations showed that detectable ceruloplasmin mRNA was present in all of the patients with Wilson's disease, including one patient with no detectable serum ceruloplasmin. However, the ceruloplasmin mRNA levels in the Wilson's disease patients were only 33% that of controls (P less than 0.001). In contrast, albumin mRNA levels in the Wilson's disease patients averaged 161% that of controls. In an attempt to better delineate the level of gene expression responsible for this decrease in ceruloplasmin mRNA, the nuclear run-on assay was used to analyze transcriptional rates. The amount of ceruloplasmin gene transcription in four Wilson's patients was decreased to 44% that of three controls. These results indicate that the diminished serum ceruloplasmin levels in patients with Wilson's disease are due at least in part to a decrease in ceruloplasmin gene transcription.

Ceruloplasmin↗

PubMed1986

Complete cDNA sequence of human preceruloplasmin.

A cDNA for human ceruloplasmin (EC 1.16.3.1) was identified in a human liver cDNA library by screening with two mixtures of synthetic oligodeoxyribonucleotides that were complementary to two regions of ceruloplasmin mRNA as predicted from the amino acid sequence of plasma ceruloplasmin. The resulting clone (phCP1) contained DNA coding for amino acid residues 202-1046 of the protein, followed by a stop codon, a 3' untranslated region of 123 base pairs, and a poly(A) tail. To isolate cDNAs encoding the 5' end of ceruloplasmin mRNA, a cDNA library was constructed in lambda gt10. The cDNA for this library was synthesized by reverse transcription of human liver poly(A)+ RNA, using random oligonucleotides as primers. When this cDNA library was screened by using a 5' fragment of phCP1 as a hybridization probe, several positive clones were identified. One of these clones (lambda hCP1) contained DNA coding for a probable signal peptide of 19 amino acid residues followed by DNA coding for residues 1-380 of plasma ceruloplasmin. Blot hybridization analysis showed that ceruloplasmin mRNA from human liver and the human hepatoma cell line HepG2 is 3700 nucleotides in size. Liver contained an additional mRNA species that is like ceruloplasmin mRNA and is 4500 nucleotides in size. Comparison of the complete nucleotide sequences of human ceruloplasmin cDNA and human clotting factor VIII cDNA showed regions of sequence homology, suggesting that these two proteins have evolved from a common ancestor.

Base Sequence↗