Expression and purification of kringle 4-type 2 of human apolipoprotein (a) in Escherichia coli

Li, Zhigao; Gambino, Roberto; Fless, Gunther M.; Copeland, Robert A.; Halfpenny, Anne J.; Scanu, Angelo M.

doi:10.1016/1046-5928(92)90017-q

Cited by 15 publications

(8 citation statements)

References 31 publications

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“…However, this pattern can also reflect a natural broadening of the peak independent of lysine binding. In support of the latter interpretation are the results on our K42 re- combinant studies showing a comparable pattern of elution that was unaffected by the presence of EACA (28). Independent verification ofthe low affinity ofrhesus Lp(a) for lysine was obtained using '25I-Lp(a).…”

Section: Resultssupporting

confidence: 74%

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Rhesus monkey lipoprotein(a) binds to lysine Sepharose and U937 monocytoid cells less efficiently than human lipoprotein(a). Evidence for the dominant role of kringle 4(37).

et al. 1993

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Rhesus lipoprotein(a) (LpIal) binds less efficiently than human Lp(a) to lysine-Sepharose and to cultured U937 cells. Studies using elastase-derived plasminogen fragments indicated that neither kringle 5 nor the protease domain of Lp(a) are required in these interactions pointing at an involvement of the K4 region. Comparative structural analyses of both the human and simian apo(a) K4 domain, together with molecular modeling studies, supported the conclusion that K437 plays a dominant role in the lysine binding function of apo(a) and that the presence of arginine 72 rather than tryptophan in this kringle can account for the functional deficiency observed with rhesus Lp(a). These in vitro results suggest that rhesus Lp(a) may be less thrombogenic than human Lp

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Section: Resultssupporting

confidence: 74%

“…this conclusion will require expression of the individual kringles and assessment of their function. This approach has been implemented by our group (28).…”

Section: Discussionmentioning

confidence: 99%

Rhesus monkey lipoprotein(a) binds to lysine Sepharose and U937 monocytoid cells less efficiently than human lipoprotein(a). Evidence for the dominant role of kringle 4(37).

et al. 1993

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“…125 I-r-apo(a) was denatured, reduced, and carboxymethylated according to Li et al 28 Briefly, !25 I-rapo(a) (150 fig) in 0.5 mol/L Tris-HCl, pH 8.0, was denatured by the addition of 6 mol/L guanidine-HCl and 50 mmol/L dithiothreitol. After incubation for 1 hour at 25°C under a nitrogen atmosphere, iodoacetic acid was added to a final concentration of 150 mmol/L, and the reactants were removed by dialysis.…”

Section: Modifications Of R-apo(a)mentioning

confidence: 99%

“…38 Consistent with these ideas is the finding that under conditions in which a 50-fold molar excess of unlabeled r-apo(a) caused 83% inhibition of 125 I-r-apo(a) degradation by foam cells, a 400-fold molar excess of a single-copy recombinant K4 peptide (cf Reference 28) caused only 21% inhibition (G.A.K., Y.L, G.M.F., and I.T., unpublished data). Although the relatively weak competitive inhibition by the recombinant peptide might be explained by its expression in a noneukaryotic system (ie, not glycosylated), the peptide is recognized by a monoclonal antibody that can distinguish native from reduced K4 2 , 28 and the recombinant kringle possesses other features that suggest that its overall conformation is normal. 28 Thus, two or more kringles in tandem may be necessary for optimal interaction with the foam cell receptor.…”

mentioning

confidence: 99%

“…Although the relatively weak competitive inhibition by the recombinant peptide might be explained by its expression in a noneukaryotic system (ie, not glycosylated), the peptide is recognized by a monoclonal antibody that can distinguish native from reduced K4 2 , 28 and the recombinant kringle possesses other features that suggest that its overall conformation is normal. 28 Thus, two or more kringles in tandem may be necessary for optimal interaction with the foam cell receptor. Further testing of this idea, including the determination of whether specific K4 sequences or interkringle sequences are involved and how many K4 sequences in tandem are necessary for receptor interaction, must await the availability of a series of recombinant kringle constructs.…”

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confidence: 99%

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Macrophage foam cell lipoprotein(a)/apoprotein(a) receptor. Cell-surface localization, dependence of induction on new protein synthesis, and ligand specificity.

Keesler

Skiba

et al. 1994

Arterioscler Thromb

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Understanding the interaction of the atherogenic lipoprotein, lipoprotein(a) [Lp(a)], with macrophages may provide important insight into the physiology and pathophysiology of this lipoprotein. We have recently shown that cholesterol loading of macrophages, such as occurs in atheroma foam cells, leads to marked upregulation of a novel receptor activity for native Lp(a) and its plasminogen-like protein component, apoprotein(a) [apo(a)]. We show here that the Lp(a)/apo(a) receptor activity on cholesterol-loaded macrophages is trypsin sensitive, indicating that a cell-surface protein is involved and that the upregulation by cholesterol loading requires new protein synthesis. Ligand studies revealed that the foam cell receptor activity recognizes Lp(a) containing both small and large isoforms of apo(a) as well as rhesus monkey Lp(a), which contains an inactive kringle-4 37 (K4 37 ) lysine-binding domain. Elastase degradation products of plasminogen did not compete for 12 Elevated plasma levels of Lp(a) occur in «=20% of the adult Caucasian population, and there appears to be an association between elevated Lp(a) levels and the occurrence of coronary atherosclerosis.1 -2 Furthermore, cholesterolfed apo(a) transgenic mice develop significantly more atherosclerosis than nontransgenic controls.3 Despite several important hypotheses and in vitro observations regarding possible roles of Lp(a) in atherosclerosis, 47 neither the mechanism of Lp(a) atherogenicity nor the normal physiological roles of the lipoprotein are definitively known. Nonetheless, the interaction of Lp(a) with macrophages is thought to be important, since cholesteryl ester-filled macrophages (foam cells) are a prominent feature of atherosclerotic lesions. 810 In fact, apo(a) has been found to colocalize with foam cells in these lesions. © 1994 American Heart Association, Inc.interaction. Consistent with these data, the degradation of 125 I-r-apo(a) was completely blocked by an anti-Lp(a) polyclonal antibody that does not cross-react with plasminogen. Furthermore, the multiple sialic residues of apo(a) are also not involved in receptor interaction, since desialylated r-apo(a) interacted with foam cells as well as native r-apo(a Previous studies have shown that cultured macrophages internalize and degrade native Lp(a) and apo(a) poorly. 1215 Recent work from our laboratory, however, revealed an Lp(a) receptor activity, different from known lipoprotein receptors, that can be induced in macrophages by cholesterol loading. 16 The interaction of Lp(a) with cholesterol-loaded macrophages is solely dependent on the apo(a) moiety of Lp(a), since lipid-free recombinant apo(a) [r-apo(a)] but not apo(a)-free Lp(a-) is recognized by the foam cell receptor activity. 16In the present study, we investigated whether a cell-surface protein is involved in the foam cell Lp(a)/ apo(a) receptor activity and whether upregulation induced by cholesterol loading requires new protein synthesis. Furthermore, we explored several key properties of ligands that are need...

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Synthesis and expression of w-conotoxin MVIIA

Cai

Chinese Peptide Symposia

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Expression and purification of kringle 4-type 2 of human apolipoprotein (a) in Escherichia coli

Cited by 15 publications

References 31 publications

Rhesus monkey lipoprotein(a) binds to lysine Sepharose and U937 monocytoid cells less efficiently than human lipoprotein(a). Evidence for the dominant role of kringle 4(37).

Rhesus monkey lipoprotein(a) binds to lysine Sepharose and U937 monocytoid cells less efficiently than human lipoprotein(a). Evidence for the dominant role of kringle 4(37).

Macrophage foam cell lipoprotein(a)/apoprotein(a) receptor. Cell-surface localization, dependence of induction on new protein synthesis, and ligand specificity.

Synthesis and expression of w-conotoxin MVIIA

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