Transgenic fish

1. Mutation in human PCSK9

• Conserved region between zebra fish and human PCSK9
• Choose the mutation
• p.S127R and D129G
• S127 and D129 highly conserved across different species in the prodomain, signifies that
the prodomain is necessary for degradation of LDLR
• s127R five fold higher affinity to LDLR compared to wild type PCSK9
• cleavage of
• PCSK9 does not undergo autocatalytic cleavage (however 20% of mutant PCSK9 undergoes
autocatalytic cleavage and in secreted)
• Remains intracellular and decreases the expression and activity of LDLR
• Segregates with FH phenotype
2. Interaction between endogenous LDLr and exogenous PCSK9 (if
present) knock out endogenous PCSK9 might be required
3. Cloning
4. Microinjection
Conserved region between zebra fish and human PCSK9

p.S127R and p.D374Y

These mutations are more potent in reducing the number of LDLR
 p.S127R
• Located in the prodomain
• Highly conserved in different species
• Segregates with phenotype
• This mutation modulates the interactions with cellular machineries that
sort LDL receptor to lysosomes
• Mutation doesn’t affect the interaction with PCSK9 (affinity same as that
of WT)
• Enhances the sorting of LDLR to lysosomes rather than affecting LDLR-
PCSK9 interaction
• Lowers autoprocessing and secretion of PCSK9 (mech.unkown) (S127
could play an important role in the recognition of the cleavage site for
autoprocessing)
• Vertical scanning mutagenesis for S127 resulted in S127R showing 4.3
fold reduction in the uptake of LDL
• the mutations confers a gain of function phenotype by
enhancing the affinity of PCSK9 for LDLR, either prior to
secretion of the protein, or after cellular internalization
of the PCSK9-LDLR complex into endosomes.
• Alternatively, it is possible that the prodomain of PCSK9
is recognized by an accessory protein or coreceptor
required for PCSK9-mediated degradation of LDLR, and
that S127R mutations strengthen this interaction,
resulting in more efficient trafficking of the PCSK9-LDLR
complex to lysosomes for degradation.
 D374Y
• Present in catalytic domain
• Has no effect in auto processing or secretion
• Has higher affinity with PCSK9 (∼25-fold lower concentration
than that of wild-type PCSK9)
• Asp374 is located where the surrounding residues of LDLR
disfavor Asp374, such as negatively charged or hydrophobic
residues. Therefore, Asp374 prefers to form an intramolecular
hydrogen bond with Ser221 of PCSK9. Mutation of Asp374 to
neutral hydrophobic residues such as tyrosine could then relieve
the unfavorable charge interactions with LDLR, thereby
strengthening the PCSK9-LDLR interaction (bonds with His306
LDLR / H306Y LDLR same effect as that of D374Y PCSK9)
• Mutant PCSK9 proteins carrying select mutations at
S127, including S127R and S127K, were more
potent in lowering cellular LDL uptake, whereas
each mutation made at Asp374 resulted in a more
potent PCSK9 protein. The combination of both
S127R and D374Y PCSK9 mutations had an additive
effect on PCSK9 function, resulting in an
approximately 70-fold increase in the potency of
PCSK9-mediated lowering of LDL uptake upon
addition of purified protein to cells
 increase PCSK9 stability

decrease PCSK9 stability

http://www.jlr.org/content/49/6/1333/F5.expansion.html
• It is important to mention that in many cell lines, PCSK9 is further
cleaved to a 53 kDa protein [9,11]. The residue that appears to be
involved in the production of this further cleaved form is Arg218
[28]. Not only is this Arg residue conserved between species, but it
is also found within an RXXR or KXXXXR sequence which is a basic
amino acid recognition site specific for furin and/or PC5/6-like
enzymes [28,33]. In fact, studies have demonstrated that
membrane bound furin and to a lesser extent the soluble PC5/6A
are capable of cleaving PCSK9 through this RXXR motif [28,33]. The
53 kDa PCSK9 form is inactive and unable to bind the prosegment
[28,33]. Whether this second cleavage of PCSK9 by furin and/or
PC5/6 occurs or is significant in vivo requires further elucidation
• D374H
• D374F