Investigations on the evolutionary conservation of PCSK9
reveal a functionally important protrusion
Jamie Cameron
1
, Øystein L. Holla
1
, Knut Erik Berge
1,
*, Mari Ann Kulseth
1
, Trine Ranheim
1
,
Trond P. Leren
1
and Jon K. Laerdahl
2
1 Medical Genetics Laboratory, Department of Medical Genetics, Rikshospitalet University Hospital, Oslo, Norway
2 Centre for Molecular Biology and Neuroscience (CMBN), Institute of Medical Microbiology, Rikshospitalet University Hospital, Oslo,
Norway
An elevated level of plasma low-density lipoprotein
(LDL) cholesterol is a major risk factor for coronary
heart disease. The key factor regulating the level of
LDL cholesterol is the cell surface LDL receptor
(LDLR) [1]. The number of LDLRs is regulated at the
transcriptional level [1] but is also post-transcription-
ally regulated by proprotein convertase subtilisin⁄ kexin
type 9 (PCSK9) [2], also known as NARC-1 [3]. Over-
expression of PCSK9 in mice leads to reduced levels of
LDLR and increased levels of LDL cholesterol [2,4,5],
whereas mice with no functional PCSK9 have

In this study, we have assembled homologs of human PCSK9 from 20 ver-
tebrates, a cephalochordate and mollusks in order to search for conserved
regions of PCSK9 that may be important for the PCSK9-mediated degra-
dation of LDLR. We found a large, conserved protrusion on the surface of
the PCSK9 catalytic domain and have performed site-directed mutagenesis
experiments for 13 residues on this protrusion. A cluster of residues that is
important for the degradation of LDLR by PCSK9 was identified. Another
cluster of residues, at the opposite end of the conserved protrusion, appears
to be involved in the physical interaction with a putative inhibitor of
PCSK9. This study identifies the residues, sequence segments and surface
patches of PCSK9 that are under strong purifying selection and provides
important information for future studies of PCSK9 mutants and for inves-
tigations on the function of this regulator of cholesterol homeostasis.
Abbreviations
CRD, cysteine-rich domain of PCSK9, i.e. the C-terminal domain; EGF-A, epidermal growth factor-like repeat A of LDLR; EST, expressed
sequence tag; LDL, low-density lipoprotein; LDLR, low-density lipoprotein receptor; PC, proprotein convertase; PCSK9, proprotein
convertase subtilisin ⁄ kexin type 9; WT, wild-type.
*[Correction added on 16 July 2008, after first online publication: the author name has been amended]
FEBS Journal 275 (2008) 4121–4133 ª 2008 The Authors Journal compilation ª 2008 FEBS 4121
LDLR is internalized by endocytosis [7,8], and bound
PCSK9 somehow disrupts the recycling of the LDLR.
As a consequence, the LDLR is transferred to the
lysosomes for degradation [7].
PCSK9 belongs to a superfamily of subtilisin-like
serine proteases and is the ninth mammalian member
identified in the proprotein convertase (PC) family [3].
The PC zymogens have an N-terminal signal sequence,
a prodomain, a catalytic subtilisin-like domain, and a
C-terminal domain [9]. They undergo autocatalytic
cleavage in the endoplasmic reticulum, but the prodo-

region of resistin [15] and is held together by three
structurally conserved disulfide bonds.
In humans, various mutations in the PCSK9 gene
have been found to cause autosomal dominant hypo-
cholesterolemia or hypercholesterolemia [4,13,17–24].
For mutations that do not affect PCSK9 folding or
secretion, these effects appear to be largely mediated
by different affinities of the mutant PCSK9s for the
LDLR [11,25]. However, another level of complexity
has been added with the recent finding that PCSK9
itself is cleaved by the PC furin, and, to a lesser extent,
by PC5 ⁄ 6A [26]. PCSK9 is cleaved between resi-
dues 218 and 219 in what has been shown to be a
structurally disordered loop on the surface of the
PCSK9 catalytic domain [10,15,16]. Furin-cleaved
PCSK9 is inactive in degrading LDLR, and naturally
occurring gain-of-function mutations such as R215H
[24], F216L and R218S [17,27] are likely to be gain-of-
function mutations due to reduced furin cleavage.
The exact mechanism by which PCSK9 binds to the
LDLR and disrupts the normal recycling of the LDLR
remains to be determined. One strategy to elucidate
the underlying mechanism is to study how mutations
in the PCSK9 gene affect the PCSK9-mediated degra-
dation of the LDLR. Candidate residues for being of
functional importance for macromolecular interactions
involving PCSK9 are those that are highly conserved
between different species, especially conserved residues
that are solvent-exposed in unbound PCSK9 and that
do not appear to be important for protein folding.

patch giving rise to mutations of the loss-of-function
type. Our data suggest that the conserved protrusion is
involved in two separate specific macromolecular inter-
actions of importance for the PCSK9-mediated degra-
dation of the LDLRs.
Conserved protrusion on PCSK9 J. Cameron et al.
4122 FEBS Journal 275 (2008) 4121–4133 ª 2008 The Authors Journal compilation ª 2008 FEBS
Results
PCSK9 has homologs in chordates and mollusks
Homologs of human PCSK9 were extracted from a
number of public databases, including the NCBI non-
redundant protein and expressed sequence tag (EST)
databases [28], uniprot [29], the ensembl resources
[30], and some sequencing project databases. Protein
sequences homologous to full-length human PCSK9
from many vertebrates were found, including primates,
rat, mouse, squirrel (Spermophilus tridecemlineatus),
and a number of other placental mammals, opossum
(Monodelphis domestica), chicken, the Carolina anole
lizard (Anolis carolinensis), frogs (Xenopus tropicalis ⁄
Xenopus laevis), and the fish species Oryzias latipes,
Danio rerio, Tetraodon nigroviridis, and Takifugu rubri-
pes (see supplementary Doc. S1). No vertebrate with
more than a single PCSK9 homolog was found.
Data from the Florida lancelet (B. floridae) sequenc-
ing project, containing both genomic and EST
sequences, indicate at least two potential homologs of
full-length PCSK9 in this organism. B. floridae is a
representative of the cephalochordates, one of the
three chordate subphyla, the other two being verte-

to be a premature stop codon in putative bovine
exon 10. Traces sequenced in both directions on the
genome are available in the NCBI Trace Archive that
supports the stop codon in exon 10. The Btau_3.1 ver-
sion of the bovine genome is a preliminary assembly
based on approximately 26 million reads and  7·
sequence coverage. Close to 95% of bovine ESTs were
contained in the assembled contigs, indicating that less
than one in 20 Bos taurus protein-coding genes are
missing in this assembly.
The above findings suggest that the region on bovine
chromosome 3 with homology to PCSK9 is a remnant
of a PCSK9 pseudogene, and that extant Bos taurus
might be lacking functional PCSK9.
Site-directed mutagenesis of residues in a
conserved protrusion on PCSK9
On the basis of a multiple sequence alignment of 18
vertebrate PCSK9 homologs, residue conservation
was mapped onto a PCSK9 structural model with the
consurf tool [31,32] (Fig. 2). Residue conservation
on the solvent-exposed PCSK9 surface is limited. The
exception is a large protrusion on the catalytic
domain with a surface area of roughly 1500 A
˚
2
(Fig. 2B). Approximately half of this protrusion, the
part closest to the prodomain, is built from the struc-
turally disordered loop Gly213–Arg218 (Fig. 2A) and
residues partially covered by this loop (Fig. 2C). Evo-
lutionarily conserved regions on protein surfaces are

retained in the endoplasmic reticulum due to abnormal
protein folding caused by disruption of the disulfide
bond bridging the residues Cys375 and Cys378.
Effect of PCSK9 mutants on the internalization
of LDL and on PCSK9 cleavage by furin
To study the effects of the 13 PCSK9 mutants on the
PCSK9-mediated degradation of the LDLR, we used
transiently transfected HepG2 cells and studied the
amount of LDL internalization by flow cytometry.
HepG2 cells transfected with WT-PCSK9, empty plas-
mid, the catalytically inactive S386A-PCSK9 plasmid
[3,23] or one of the two gain-of-function plasmids,
S127R-PCSK9 and D374Y-PCSK9 [23], were used as
controls (Fig. 4). Internalization of LDL by cells
A
B
C
Fig. 1. Multiple sequence alignments of human PCSK9 homologs from vertebrates, a cephalochordate (B. floridae) and the mollusks Aplysia
and Biomphalaria, showing the signal sequence and N-terminus of the prodomain (A), two segments of the catalytic domain (B), and the full
CRD, the C-terminal domain (C). Conserved residues are indicated by numbering referring to human PCSK9. The catalytic triad residues are
marked with an asterisk. The full alignment and sequence data are given in supplementary Figs S1, S2 and supplementary Table S1.
Conserved protrusion on PCSK9 J. Cameron et al.
4124 FEBS Journal 275 (2008) 4121–4133 ª 2008 The Authors Journal compilation ª 2008 FEBS
expressing these control plasmids was comparable to
previous findings [23,24].
Cells expressing the two mutants C375A-PCSK9
and C378A-PCSK9 internalized 19% and 14% more
LDL, respectively, than cells expressing WT-PCSK9.
Thus, as expected for PCSK9 mutants that are secreted
at markedly reduced levels, the two mutants present as

internalized less LDL than cells expressing WT-PCSK9
(Fig. 4). The amounts of LDL internalized by cells
expressing these mutants were in the same range or
lower than in cells expressing the gain-of-function
mutant S127R-PCSK9. Thus, we consider these
mutants to be gain-of-function mutants.
The four loss-of-function mutants involving Arg194,
Asp238, Thr377 or Phe379 are located close together
on the conserved protrusion (Fig. 2C). Four gain-of-
function mutants, involving Gln190, Lys222, Asp374
and Ser376, are located together in a separate region
between the loss-of-function patch and the disordered
loop Gly213–Arg218. The two remaining gain-of-func-
tion mutants, involving Ser153 and Asp204, are
located on opposing edges of the conserved protrusion
(Fig. 2C).
Five of the six gain-of-function mutant residues are
clustered in the vicinity of the disordered loop consist-
ing of residues Gly213–Arg218 (Fig. 2C). This loop
contains the furin cleavage site RFHR
218
[26], and
cleavage by furin at this site results in PCSK9 that is
inactive in degrading the LDLR [26]. To determine
whether the gain-of-function mutants had reduced
furin cleavage, the amounts of furin-cleaved PCSK9 in
the media of HEK293 cells transiently transfected with
the different PCSK9 plasmids were determined by wes-
tern blot analysis. HEK293 cells were chosen for these
analyses because truncated PCSK9 due to cleavage by

media of HepG2 cells and HEK293 cells transfected
with the R194A-PCSK9 plasmid or the D204A-PCSK9
plasmid. However, the corresponding uncleaved pro-
PCSK9 (Fig. 3) and the furin-cleaved PCSK9 (Fig. 5)
appeared to migrate normally. To study whether the
abnormal migration of the mature forms of R194A-
PCSK9 and D204A-PCSK9 was due to altered auto-
catalytic cleavage, western blot analyses of media from
transfected HepG2 cells were performed using an anti-
body against the prodomain of PCSK9. The prodo-
mains of R194A-PCSK9 and D204A-PCSK9 migrated
normally (Fig. 6). Thus, the two mutants were autocat-
alytically cleaved in a normal fashion.
To study whether the abnormal migration of
mature, cleaved R194A-PCSK9 and D204A-PCSK9
was due to abnormal glycosylation, the sensitivities of
R194A-PCSK9 and D204A-PCSK9 to an enzyme mix
designed to remove all sugars were determined in cell
lysates of transiently transfected HepG2 cells. The
results showed that the differences in the migration of
mature PCSK9 remained after the enzyme treatment
(Fig. 7). Thus, an abnormal post-translational modifi-
cation other than glycosylation appears to be respon-
sible for the abnormal migration of R194A-PCSK9
and D204A-PCSK9.
Discussion
Vertebrate genome sequencing projects are currently
supplying the research community with sequence data
from a large number of species that have varying evo-
lutionary relationships with humans. The data from

plasmids containing R194A-PCSK9 or D204A-PCSK9 with or with-
out prior treatment with the Glycoprotein Deglycosylation Kit. A
horizontal dotted line is included to show that all the mature
PCSK9s after deglycosylation have increased mobility due to degly-
cosylation.
J. Cameron et al. Conserved protrusion on PCSK9
FEBS Journal 275 (2008) 4121–4133 ª 2008 The Authors Journal compilation ª 2008 FEBS 4127
LDL as compared to WT-PCSK9 (Fig. 4). Only one
of these residues, Asp374, has previously been associ-
ated with hypercholesterolemia in human populations
[18,19,33].
Previous studies have described a number of natu-
rally occurring loss-of-function mutations in PCSK9
that result in proteins that are not autocatalytically
cleaved and ⁄ or not folded properly [4,10,13,23,24].
Apart from the previously studied active site mutant
S386A-PCSK9 [23], the only mutants of the present
study that clearly have impaired secretion are C375A-
PCSK9 and C378A-PCSK9 (Fig. 3). Both residues are
absolutely conserved in all chordates (Fig. 1B and sup-
plementary Fig. S1), demonstrating their importance
for the formation of a disulfide bridge stabilizing the
conformation and overall shape of the conserved
protrusion (Fig. 2).
Four of the mutants, R194A-PCSK9, D238A-
PCSK9, T377A-PCSK9, and F379A-PCSK9, were
secreted in a similar fashion as WT-PCSK9 (Fig. 3),
but nevertheless present as loss-of-function mutants
(Fig. 4). The residues involved are located close
together on the conserved protrusion, at the far end

with the part of the conserved protrusion associated
with loss-of-function mutants in the present study. Our
findings that mutations R194A, D238A, T377A and
F379A were loss-of-function mutations are in agree-
ment with the notion that they diminish the binding of
PCSK9 to EGF-A.
The crystal structure obtained by Kwon et al. [34]
shows that the N-terminal amine of mature PCSK9
Ser153 forms a salt bridge with a residue in EGF-A,
but that the Ser153 side-chain does not directly contact
the binding partner. Correspondingly, our results
showed that S153A-PCSK9 is not a loss-of-function
mutant. Instead, S153A-PCSK9 appears to lead to
decreased internalization of LDL as compared to WT-
PCSK9. One might speculate that this could be due to
a slight change in the ability of residue 153 to form a
salt bridge to EGF-A, e.g. through an inductive effect.
All the other residues that were associated with
gain-of-function mutations in the present study were
located either between the EGF-A binding patch and
the disordered loop Gly213–Arg218 (Gln190, Lys222,
Asp374, and Ser376), or between the disordered loop
and the prodomain (Asp204) (Fig. 2C). These residues
are under selective pressure, with Asp204 and Asp374
being conserved in all vertebrates. Lys222 is conserved
in nonfish vertebrates, whereas the conservation of
Ser376 appears to be slightly lower (Fig. 1B and sup-
plementary Fig. S1). Mutations of the disordered loop
residues, Arg215 [24], Phe216, and Arg218 [26], located
in this part of the conserved protrusion, have previ-

romolecule is interacting in a specific manner with the
relevant part of the conserved protrusion of PCSK9.
This macromolecule may be competing with EGF-A
binding or may inhibit the PCSK9-mediated degrada-
tion of the LDLR by another mechanism. Fan et al.
[35] have recently suggested that multimerization of
PCSK9 is important for its LDLR-regulating activity.
They found that mutation of Asp374, a residue in the
conserved protrusion, affected PCSK9 self-association.
It is, however, not obvious that PCSK9 self-association
is important in vivo when PCSK9 is secreted at low
concentrations. Previous studies have found no indica-
tions of multimerization for mature PCSK9 [3].
Earlier studies have shown that the naturally occur-
ring mutant D374Y-PCSK9 binds LDLR more effi-
ciently than WT-PCSK9 [8,10,25], and Kwon et al. [34]
suggested that this was due to an additional hydrogen
bond between PCSK9 Tyr374 and His306 of the EGF-
A. We now show that D374A-PCSK9, which results in
a residue Ala374 that clearly cannot form any hydro-
gen bonds with its side-chain, is also a gain-of-function
mutant, although it is only half as potent as D374Y-
PCSK9. This may indicate that the naturally occurring
D374Y-PCSK9 is a gain-of-function mutant due to two
different mechanisms: one is to strengthen the interac-
tion between PCSK9 and EGF-A, and the other is to
disrupt the binding to PCSK9 of a putative inhibitory
macromolecule.
The sequence data that were gathered for the present
study reveal interesting phylogenetic relationships in

homologs (Fig. 1 and supplementary Figs S1 and S2)
clearly show the catalytic domain to be more con-
served than the CRD. This is also the case for PCSK9
conservation within the group of primates [36]. Resi-
due identities between human and opossum are 76%
and 53% for the catalytic domain and CRD, respec-
tively. The prodomain is also fairly well conserved,
apart from the structurally disordered region compris-
ing the N-terminal 30 residues (Fig. 1A). This segment
is very rich in acidic residues, with seven of 10 N-ter-
minal residues of human PCSK9 being Asp or Glu.
This is immediately followed by five small aliphatic
residues and a segment with five more acidic residues.
The N-terminal region of the prodomain will clearly
interact strongly and nonspecifically with a positively
charged moiety. The signal sequence is not conserved,
except for a Leu-rich segment.
The three catalytic residues are absolutely conserved
in all PCSK9 homologs (Fig. 1B), as is the last residue
of the prodomain, Gln152, supporting the notion that
these residues are essential for autocatalysis and effi-
cient secretion of PCSK9. The 18 Cys residues of the
PCSK9 CRD are conserved in all chordates, as well as
in the mollusk CRDs (Fig. 1C and supplementary
Fig. S2). This clearly demonstrates that the nine disul-
fide bridges covalently stabilizing the three modules of
this domain are essential for its processing and func-
tion. The CRD also contains a number of conserved
Ser, Thr and small aliphatic residues. These are mainly
located deep in the structure, and are most likely

[13,26]. It is conserved in placental mammals only. The
corresponding residue is not likely to be glycosylated
in other vertebrates.
In conclusion, there is a single, large, evolutionarily
conserved protrusion on the surface of the catalytic
domain of PCSK9. The lack of other residue conserva-
tion on the PCSK9 surface makes it less likely that
there are other parts of PCSK9 that interact with high
specificity with other macromolecules as part of the
PCSK9-mediated degradation of the LDLR. A cluster
of residues on the conserved protrusion is involved in
the binding of PCSK9 to the EGF-A domain of the
LDLR, and mutations of these residues lead to loss of
function, as found in our study and in the study of
Kwon et al. [34]. The part of the protrusion located
around the disordered loop Gly213–Arg218 contains a
number of conserved residues for which site-directed
mutagenesis produced gain-of-function mutants. These
residues appear to be involved in some form of inhibi-
tion of the PCSK9-mediated degradation of the
LDLR. However, our data do not clearly support a
model that solely involves reduced cleavage by furin.
Thus, further studies are needed to clarify whether
these residues are involved in the binding of a different
macromolecule that inhibits the degradation of the
LDLR by PCSK9.
Experimental procedures
Data collection and bioinformatics analysis
Database resources provided by the NCBI [28], uniprot
[29], the ensembl project [30], the DOE Joint Genome

Carlsbad, CA, USA), in a humidified atmosphere (37 °C,
5% CO
2
).
Mutagenesis, cloning and expression of PCSK9
Mutations S153A, Q190A, R194A, D204A, K222A,
R237A, D238A, D374A, C375A, S376A, T377A, C378A or
F379A were introduced into a pCMV–PCSK9–FLAG plas-
mid kindly provided by J. D. Horton (University of Texas
Southwestern Medical Center, Dallas, TX, USA), using
QuickChange XL Mutagenesis Kit (Stratagene, La Jolla,
CA, USA) according to the manufacturer’s instructions.
The primer sequences used for the mutagenesis are given in
supplementary Table S2. The resulting mutant plasmids are
referred to as S153A-PCSK9, Q190A-PCSK9, R194A-
PCSK9, D204A-PCSK9, K222A-PCSK9, R237A-PCSK9,
D238A-PCSK9, D374A-PCSK9, C375A-PCSK9, S376A-
PCSK9, T377A-PCSK9, C378A-PCSK9, and F379A-
PCSK9. The integrity of each plasmid was confirmed by
DNA sequencing. An empty plasmid, pcDNA3.1 ⁄ myc his-c
(Invitrogen), as well as four previously published mutant
PCSK9 plasmids containing mutations S386A, S127R,
R215H or D374Y [23,24], were used as controls in the
transfection experiments together with WT-PCSK9 plasmid.
Transient transfections of HepG2 cells and HEK293 cells
with WT-PCSK9 plasmid or mutant PCSK9 plasmids were
performed as described by Cameron et al. [24].
Western blot analysis of transfected HepG2 and
HEK293 cells
Western blot analyses of cell lysates and culture media of

manufacturer’s instructions. The treated lysates were sub-
jected to western blot analysis using the antibody to
PCSK9 directed at residues 490–502, as previously
described [23].
Acknowledgements
This work was supported by the Research Council of
Norway.
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Supplementary material
The following supplementary material is available
online:
Doc. S1. Supplementary materials and methods:
sequence data collection.
Fig. S1. Multiple sequence alignment of the signal
sequence, the prodomain and the catalytic domain of
PCSK9 homologs.
Fig. S2. Multiple sequence alignment of the C-terminal
domain for PCSK9 homologs.
Table S1. Sequence data for PCSK9 homologs.
Table S2. Primer sequences used to generate mutant
PCSK9 plasmids.