groups ( 21 ). As expected from B4GALT1’s
role in processing N-linked carbohydrate, all
samples had normal profiles for ApoCIII
O-linked carbohydrate (table S6). All samples
also had normal mono-oligosaccharide/di-
oligosaccharide transferrin and a-oligosaccharide/
di-oligosaccharide transferrin ratios. However,
whereas all eight^352 Asn homozygotes had
normal tri-sialo/di-oligosaccharide transferrin
ratios, this ratio in all^352 Ser homozygotes
was abnormal (P=9.1×10–^10 ,ttest) (fig. S4
and table S6). Because transferrin is most-
ly tetrasialylated ( 33 , 34 ), this increase in
trisialylated transferrin reflects a paucity of
tetrasialylated molecules and indicates that
the^352 Ser allele is associated with increased
levels of carbohydrate-deficient transferrin,
likely because of decreased enzymatic activ-
ity of B4GALT1.
To determine whether the lower levels of
sialylation only affect transferrin or if they
also affect other glycoproteins, N-linked glycan
profiling was performed using plasma samples
from 12 pairs of matched opposite homozygotes
for all plasma glycoproteins and selected
specific plasma glycoproteins: apolipoprotein
B100 (ApoB100), fibrinogen, and immuno-
globulin G (IgG) ( 21 ). There was significantly
lower galactosylation and sialylation (P< 0.0001,
all byttest) for global glycoproteins, ApoB100,
fibrinogen, and IgG among^352 Ser homozygotes
compared with^352 Asn homozygotes. There
was no difference in fucosylation (Fig. 3, A to
C), which is consistent with the role of
B4GALT1 in adding galactose moieties that
then are capped by sialic acid while having no
role in the addition of fucose ( 35 ). Glycans
released from all plasma glycoproteins of
(^352) Ser homozygotes had increased levels of
incompletely synthesized oligosaccharides, as
evidenced by increased percentages of glycans
devoid of galactoses and sialic acids (G0F,P=
5.4 × 10–^11 ) and glycans with only one galactose
and one sialic acid (G1S1,P=5.1×10–^16 ). Re-
ciprocally,^352 Ser homozygotes had significant-
ly lower levels of glycans with two galactose and
two sialic acid residues (G2S2,P= 2.6 × 10–^8 )
(table S7). The results for plasma ApoB100,
fibrinogen, and IgG were similar in that serum
from^352 Ser homozygotes had significantly in-
creased levels of glycans deficient in galactose
and sialic acid moieties and decreased levels of
more mature glycans (tables S8 to S10).
Furthermore, in these same plasma samples,
(^352) Ser homozygotes had lower levels of ApoB100
(P= 0.0075) and increased IgG (P= 0.0004)
compared with^352 Asn homozygotes (fig. S5,
A and B). Fibrinogen also trended downward
(P= 0.06) (fig. S5C), consistent with the larger
sample size in the initial discovery cohort. In
summary, the^352 Ser allele is associated with
significantly increased levels of incompletely
synthesized N-linked glycans on glycoproteins,
thus indicating defective protein glycosylation.
Altered levels of these proteins may be second-
ary to the observed defects in protein glyco-
sylation, which could affect protein synthesis,
stability, or turnover.
B4GALT1p.Asn352Ser causes reduced
enzymatic activity
To compare the in vitro enzymatic activities
of wild-type (^352 Asn) and mutant (^352 Ser)
B4GALT1 protein, we transiently transfected
COS-7 cells, which express very low endoge-
nous levels of B4GALT1, with complementary
DNAs (cDNAs) encoding myc-FLAG epitope-
tagged versions of the proteins ( 21 ). We immu-
noprecipitated with anti-FLAG antibody and
measured B4GALT1 activities in the immune
complexes (fig. S6). Compared with^352 Asn
B4GALT1,^352 Ser B4GALT1 showed, on average,
a 50% decrease in galactosyltransferase specific
activity (Fig. 4A).
As a complementary approach, we synthe-
sized recombinant human^352 Asn and^352 Ser
B4GALT1 protein to test the reduction rate
of the substrate UDP-Gal incubated with each
protein ( 21 ). Recombinant^352 Ser B4GALT1
showed a 2.5-fold decrease of substrate con-
sumption compared with the wild-type pro-
tein (P= 1.3 × 10–^7 ,ttest), indicating a decrease
in the enzymatic activity of^352 Ser B4GALT1
(Fig. 4B).
B4galt1 Asn353Ser knock-in mouse model
phenocopies the human LDL-C and
fibrinogen phenotype
HumanB4GALT1and its mouse ortholog
share 92% similarity of their coding sequence,
and the^352 Asn residue (corresponding to^353 Asn
in the murine protein sequence) is conserved
between the two species (fig. S7). To further
validate the findings from the human studies,
we generatedB4galt1 353Sergermline knock-
in mice ( 21 ), which were born at Mendelian
frequencies, survived perinatally, and showed
normal postnatal development without appar-
ent behavioral or visible physical abnormal-
ities. On a standard chow diet,B4galt1^353 Ser
mice showed significantly lower body weights
compared with wild-type mice [average 10%
SCIENCEscience.org 3 DECEMBER 2021•VOL 374 ISSUE 6572 1225
Fig. 6. Liver-specificB4galt1knockdown decreases circulating levels of LDL-C in adult mice.
(AtoC) Percentage of editing ofB4galt1genomic [(A) and (B)] and cDNA (C) sequence in liver. The number
of reads containingB4galt1INDELs was compared with the number of reads withB4galt1wild-type sequence.
(D)B4galt1total mRNA levels in liver by TaqMan analysis. The expression ofB4galt1was calculated
relative toGapdh. Values represent the mean of four technical replicates per condition ± SD. (E) LDL-C levels
were lower in the livers ofB4galt1-knockdown mice compared with controls.Pvalues are based onFtest.
Data are represented as mean ± SD (n= 6/group).
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