Monica Kong Beltran

Publications (2)8.77 Total impact

• Article: Furin-cleaved PCSK9 is active and modulates LDL receptor and serum cholesterol levels.

  Michael T Lipari, Wei Li, Paul Moran, Monica Kong-Beltran, Tao Sai, Joyce Lai, S Jack Lin, Ganesh Kolumam, Jose Zavala-Solorio, Anita Izrael-Tomasevic, David Arnott, Jianyong Wang, Andrew S Peterson, Daniel Kirchhofer
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  ABSTRACT: Proprotein convertase subtilisin/kexin 9 (PCSK9) regulates plasma LDL-c levels by regulating the degradation of LDL receptors. Another proprotein convertase, furin, cleaves PCSK9 at Arg218-Gln219 in the surface-exposed '218-loop'. This cleaved form circulates in blood along with the intact form, albeit at lower concentrations. To gain a better understanding of how cleavage affects PCSK9 function we produced recombinant furin-cleaved PCSK9 using antibody Ab-3D5, which binds the intact but not the cleaved '218-loop'. Using Ab-3D5 we also produced highly purified hepsin-cleaved PCSK9. Hepsin cleaves PCSK9 at Arg218-Gln219 more efficiently than furin, but also cleaves at Arg215-Phe216. Further analysis by size exclusion chromatography and mass spectrometry indicated that furin and hepsin produced an internal cleavage in the '218-loop' without the loss of the N-terminal segment (Ser153-Arg218), which remained attached to the catalytic domain. Both furin- and hepsin-cleaved PCSK9 bound to LDL receptor with only 2-fold reduced affinity compared to intact PCSK9. Moreover, they reduced LDL receptor levels in HepG2 cells and in mouse liver with only moderately lower activity than intact PCSK9, consistent with the binding data. Single injection into mice of furin-cleaved PCSK9 resulted in significantly increased serum cholesterol levels, approaching the increase by intact PCSK9. These findings indicate that circulating furin-cleaved PCSK9 is able to regulate LDL receptor and serum cholesterol levels, although somewhat less efficiently than intact PCSK9. Therapeutic anti-PCSK9 approaches that neutralize both forms should be the most effective in preserving LDL receptors and in lowering plasma LDL-c.
  Journal of Biological Chemistry 11/2012; · 4.77 Impact Factor
• Article: Calcium-independent inhibition of PCSK9 by affinity-improved variants of the LDL receptor EGF(A) domain.

  Yingnan Zhang, Lijuan Zhou, Monica Kong-Beltran, Wei Li, Paul Moran, Jianyong Wang, Clifford Quan, Jeffrey Tom, Ganesh Kolumam, J Michael Elliott, Nicholas J Skelton, Andrew S Peterson, Daniel Kirchhofer
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  ABSTRACT: LDL (low-density lipoprotein) receptor (LDLR) binds to its negative regulator proprotein convertase subtilisin/kexin type 9 (PCSK9) through the first EGF (epidermal growth factor-like) domain [EGF(A)]. The isolated EGF(A) domain is a poor antagonist due to its low affinity for PCSK9. To improve binding affinity, we used a phage display approach by randomizing seven PCSK9 contact residues of EGF(A), including the Ca(2+)-coordinating Asp310. The library was panned in Ca(2+)-free solution, and 26 unique clones that bind to PCSK9 were identified. Four selected variants demonstrated improved inhibitory activities in a PCSK9-LDLR competition binding ELISA. The Fc fusion protein of variant EGF66 bound to PCSK9 with a K(d) value of 71 nM versus 935 nM of wild type [EGF(A)-Fc] and showed significantly improved potency in inhibiting LDLR degradation in vitro and in vivo. The five mutations in EGF66 could be modeled in the EGF(A) structure without perturbation of the EGF domain fold, and their contribution to affinity improvement could be rationalized. The most intriguing change was the substitution of the Ca(2+)-coordinating Asp310 by a Lys residue, whose side-chain amine may have functionally replaced Ca(2+). EGF66-Fc and other EGF variants having the Asp310Lys change bound to PCSK9 in a Ca(2+)-independent fashion. The findings indicate that randomization of an important Ca(2+)-chelating residue in conjunction with "selection pressure" applied by Ca(2+)-free phage selection conditions can yield variants with an alternatively stabilized Ca(2+) loop and with increased binding affinities. This approach may provide a new paradigm for the use of diversity libraries to improve affinities of members of the Ca(2+)-binding EGF domain subfamily.
  Journal of Molecular Biology 06/2012; 422(5):685-96. · 4.00 Impact Factor