Cys4057 of apolipoprotein(a) is essential for lipoprotein(a) assembly.

Brunner, Christoph; Kraft, H. G.; Utermann, Gerd; Müller, Hans‐Joachim

doi:10.1073/pnas.90.24.11643

Cited by 148 publications

(137 citation statements)

References 33 publications

(19 reference statements)

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“…r-Apo(a)-Arg had a drastically decreased affinity to bind to LDL. Similar results were obtained in two other studies in which Cys-4057 was replaced by Ser or Gly (Koschinsky et al, 1993;Brunner et al, 1993). Small amounts…”

supporting

confidence: 88%

High-Level Expression of Various Apolipoprotein (a) Isoforms by "Transferrinfection": The Role of Kringle IV Sequences in the Extracellular Association with Low-Density Lipoprotein

Frank

K²,

Durovic³

et al. 1994

Biochemistry

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Characterization of the assembly of lipoprotein(a) [Lp(a)] is of fundamental importance to understanding the biosynthesis and metabolism of this atherogenic lipoprotein. Since no established cell lines exist that express Lp(a) or apolipoprotein(a) [apo(a)], a "transferrinfection" system for apo(a) was developed utilizing adenovirus receptor-and transferrin receptor-mediated DNA uptake into cells. Using this method, different apo(a) cDNA constructions of variable length, due to the presence of 3, 5, 7,9, 15, or 18 internal kringle IV sequences, were expressed in cos-7 cells or C H O cells. All constructions contained kringle IV-36, which includes the only unpaired cysteine residue (Cys-4057) in apo(a). r-Apo(a) was synthesized as a precursor and secreted as mature apolipoprotein into the medium. When medium containing r-apo(a) with 9, 15, or 18 kringle IV repeats was mixed with normal human plasma LDL, stable complexes formed that had a bouyant density typical of Lp(a). Association was substantially decreased if Cys-4057 on r-apo(a) was replaced by Arg by site-directed mutagenesis or if Cys-4057 was chemically modified. Lack of association was also observed with r-apo(a) containing only 3, 5, or 7 kringle IV repeats without "unique kringle IV Sequences", although Cys-4057 was present in all of these constructions. Synthesis and secretion of r-apo(a) was not dependent on its sialic acid content. r-Apo(a) was expressed even more efficiently in sialylation-defective C H O cells than in wild-type C H O cells. In transfected C H O cells defective in the addition of N-acetylglucosamine, apo(a) secretion was found to be decreased by 50%. Extracellular association with LDL was not affected by the carbohydrate moiety of r-apo(a), indicating a protein-protein interaction between r-apo(a) and apoB. These results show that, besides kringle IV-36, other kringle IV sequences are necessary for the extracellular association of r-apo(a) with LDL. Changes in the carbohydrate moiety of apo(a), however, do not affect complex formation.Lipoprotein(a) [Lp(a)] is currently the subject of intensive investigation because elevated plasma concentrations of Lp-(a) represent an independent risk factor for myocardial infarction and stroke (Scanu & Fless, 1990;Utermann, 1989Utermann, , 1990Edelberg & Pizzo, 1991). Despite its clinical importance, the physiological function of Lp (a)

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supporting

confidence: 88%

High-Level Expression of Various Apolipoprotein (a) Isoforms by "Transferrinfection": The Role of Kringle IV Sequences in the Extracellular Association with Low-Density Lipoprotein

Frank

K²,

Durovic³

et al. 1994

Biochemistry

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“…To determine the role of this Cys in the Lp(a) assembly, this amino acid was replaced by in vitro mutagenesis with Ser or Arg [31,32]. The capacity of the mutated r-apo(a) to associate with LDL was drastically reduced but not totally abolished.…”

Section: Assembly Of Lp(a): the Role Of Apo(a) Structurementioning

confidence: 99%

The assembly of lipoprotein Lp(a)

Frank¹,

Durovic²,

Kostner³

1996

Eur J Clin Investigation

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“…DNA fragments of various lengths of the cox-2 promoter regions were prepared by PCR using the above plasmid as a template and Pyrobest TM DNA polymerase (Takara, Kyoto, Japan). Mutated fragments were prepared by two-stage bridge PCR, using mutated primers previously reported by Brunner et al (32). These fragments were inserted into pGL2 basic vectors (Promega, Madison, WI), by using a Ligation kit version II® (Takara).…”

Section: Methodsmentioning

confidence: 99%

“…Although the CREB binding to its CRE site was unaffected by the shear stress, phosphorylation of CREB was induced by the shear stress. The activated function of CREB is also modulated by phosphorylation by several kinases (32,44) and is mediated by coactivators such as CBP and p300 (45). Thus, shear stressinduced kinase activities could have induced the phosphorylation of the above transcription factors, by which the COX-2 transcription could be activated.…”

Section: Figmentioning

confidence: 99%

Fluid Shear Stress-induced Cyclooxygenase-2 Expression Is Mediated by C/EBP β, cAMP-response Element-binding Protein, and AP-1 in Osteoblastic MC3T3-E1 Cells

Ogasawara

Arakawa

Kaneda

et al. 2001

Journal of Biological Chemistry

145

104

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Mechanical loading is crucial for maintenance of bone integrity and architecture, and prostaglandins are an important mediator of mechanosensing. Cyclooxygenase-2 (COX-2), an inducible isoform of prostaglandin G/H synthase, is induced by mechanical loading-derived fluid shear stress in bone-forming cells such as osteoblasts and osteocytes. In this study, we investigated transcription factor and transcriptional regulatory elements responsible for the shear stress-induced COX-2 expression in osteoblastic MC3T3-E1 cells. When the cells were transfected with luciferase-reporter plasmids including the 5-flanking region of the murine cox-2 gene, the fluid shear stress increased the luciferase activities, consistent with the induction of COX-2 mRNA and protein expression. Deletion analysis of the promoter region revealed that the shear stress-induced luciferase responses were regulated by two regions, ؊172 to ؊100 base pair (bp) and ؊79 to ؊46 bp, of the cox-2 promoter, in which putative cis-elements of C/EBP ␤, AP-1, cAMP-response element-binding protein (CREB), and E box are included. Mutation of sites of C/EBP ␤, AP-1, and/or cAMP-response element decreased the shear stress-induced luciferase activities, whereas mutation of the E box did not affect the responses. In an electrophoretic mobility shift assay, shear stress enhanced nuclear extract binding to double-stranded oligonucleotide probes containing C/EBP ␤ and AP-1-binding motifs, and the bands of the complexes were supershifted by the addition of antibody specific for each regulator. Although the binding activity of CREB toward its probe was unaffected by shear stress, the phosphorylation of CREB was enhanced by the stress. These data suggest that C/EBP ␤, AP-1, and CREB play crucial roles in the shear stress-induced cox-2 expression in osteoblasts.Mechanical loading applied to the skeleton is crucial to the development and maintenance of bone integrity and architecture. A decrease in the mechanical loading due to prolonged immobilization or weightlessness in space reduces the bone formation rate, resulting in bone loss (1-3). On the other hand, an increase in mechanical loading causes a gain in bone density (4, 5). Thus, bone tissue is sensitive to mechanical stimulation. Mechanical loading on bone generates extracellular matrix deformation and fluid flow, and the mechanical stimuli are translated to mechanical signals such as mechanical strain and fluid shear stress, respectively (6). Evidence obtained from in vitro studies indicates that osteocytes embedded in the lacunae/ canaliculi system and osteoblasts and bone cells lining the bone surface are mechanosensors that detect load-derived mechanical stimuli (7,8). By these bone-forming cells, the mechanical stimuli are translated into cellular signaling factors.Mechanical stress induces the expressions of several kinds of proteins in bone-forming cells such as insulin-like growth factor-I and -II, transforming growth factor-␤, osteocalcin, osteopontin, c-Fos, nitric-oxide synthase, and cyclooxygenase-2 (COX-...

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Cys4057 of apolipoprotein(a) is essential for lipoprotein(a) assembly.

Cited by 148 publications

References 33 publications

High-Level Expression of Various Apolipoprotein (a) Isoforms by "Transferrinfection": The Role of Kringle IV Sequences in the Extracellular Association with Low-Density Lipoprotein

High-Level Expression of Various Apolipoprotein (a) Isoforms by "Transferrinfection": The Role of Kringle IV Sequences in the Extracellular Association with Low-Density Lipoprotein

The assembly of lipoprotein Lp(a)

Fluid Shear Stress-induced Cyclooxygenase-2 Expression Is Mediated by C/EBP β, cAMP-response Element-binding Protein, and AP-1 in Osteoblastic MC3T3-E1 Cells

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