Human bile salt-stimulated lipase has a high frequency of sizevariation due to a hypervariable region in exon 11 Susanne Lindquist1, Lars BlaÈckberg2and Olle Hernell1 Departments of 1 Cl
Trang 1Human bile salt-stimulated lipase has a high frequency of size
variation due to a hypervariable region in exon 11
Susanne Lindquist1, Lars BlaÈckberg2and Olle Hernell1
Departments of 1 Clinical Sciences, Pediatrics and 2 Medical Biosciences, Medical Biochemistry, UmeaÊ University, Sweden
The apparent molecular mass of human milk bile
salt-stimulated lipase (BSSL) varies between mothers The
molecular basis for this is unknown, but indirect evidence
has suggested the dierences to reside in a region of
repeats located in the C-terminal part of the protein We
here report that a polymorphism within exon 11 of the
BSSL gene is the explanation for the molecular variants of
BSSL found in milk By Southern blot hybridization we
analyzed the BSSL gene from mothers known to have
BSSL of dierent molecular masses in their milk
A polymorphism was found within exon 11, previously
shown to consist of 16 near identical repeats of 33 bp each
We detected deletions or, in one case, an insertion
corres-ponding to the variation in molecular mass of the BSSL protein found in milk from the respective woman Fur-thermore, we found that 56%, out of 295 individuals studied, carry deletions or insertions within exon 11 in one
or both alleles of the BSSL gene Hence, this is a hyper-variable region and the current understanding that exon 11
in the human BSSL gene encodes 16 repeats is an over-simpli®cation and needs to be revisited Natural variation
in the molecular mass of BSSL may have clinical impli-cations
Keywords: BSSL; lipase; human milk; repeats; poly-morphism
Bile salt-stimulated lipase (BSSL) or carboxyl ester lipase
is a digestive enzyme secreted from exocrine pancreas in
all species examined BSSL has a broad substrate
speci®city and contributes to the hydrolysis of dietary
mono-, di-, and tri-acylglycerols and is responsible for
digestion of fat-soluble vitamin esters and cholesterol
esters in the small intestine In some species, including
humans, the gene is also expressed in the lactating
mammary gland and the resulting protein is a constituent
of the milk [1,2] Milk BSSL is a major reason why
breast-fed infants digest and absorb fat more ef®ciently
than formula-fed infants [3] Moreover, BSSL has been
detected in low, but signi®cant, levels in serum [4] The
function of BSSL in serum is unknown, but it has been
suggested to in¯uence the level of serum cholesterol [5,6]
Deduced from the cDNA sequence, the human BSSL
protein consists of 722 amino acids with a predicted
molecular mass of 76 kDa [7±10] The protein is, however,
abundantly glycosylated and the apparent molecular mass
on SDS/PAGE has been estimated to 120±140 kDa
[11,12] Human BSSL has a unique primary structure as
compared to other mammalian lipases The N-terminal
part of the protein shows striking homology to
acetylcho-linesterase and some other esterases [7] The C-terminal
part has been reported to consist of a unique structure
with 16 proline-rich, O-glycosylated repeats of 11 amino-acid residues each [7±10] The biological function of the repeated region is not fully understood It has been shown that the repeats protect the protein from denaturation by acid and from proteolysis by pepsin or pancreatic prote-ases in vitro [13,14] It has also been shown that the O-glycosylation of the repeated sequences is important for secretion of rat pancreatic BSSL [15] On the other hand,
we and others have shown that the repeats are completely dispensable for the typical functional properties of BSSL, i.e catalytic activity, bile-salt activation, heparin binding, heat stability, stability at low pH and resistance to proteolytic inactivation [16±18]
The BSSL protein is well conserved between species, but the number of proline-rich repeats varies, from three in cow and mouse [19,20] to 16 in the human [7±10] The salmon enzyme seems to be completely devoid of repeats [21]
The human gene encoding BSSL spans 9.8 kb and consists of 11 exons [22] The gene has been mapped to chromosome 9q34-qter and the BSSL locus was shown to exhibit a high degree of polymorphism [23] A correlation between BSSL genotype and serum cholesterol levels has been proposed [24,25] but to our knowledge, the polymor-phism in the BSSL gene has not been further characterized until now
The carboxyl ester lipase like (CELL) gene is a ubiqui-tously transcribed pseudogene for BSSL [22,26] The sequence of the CELL gene is in some parts identical to BSSL, i.e exons 1, 8 and 9, whereas there are some major differences in other parts A 4.8-kb fragment, spanning exons 2±7 in the BSSL gene, is deleted in CELL There are also several base substitutions within exons 10 and 11
A region in exon 11, encoding the proline-rich repeats, differs between BSSL and CELL Human BSSL has previously been shown to carry 16 repeats, although in this
Correspondence to S Lindquist, Department of Clinical Sciences,
Pediatrics, UmeaÊ University, SE-901 85 UmeaÊ, Sweden.
Fax: + 46 90 123728, Tel.: + 46 90 7852128,
E-mail: susanne.lindquist@pediatri.umu.se
Abbreviations: BSSL, bile salt-stimulated lipase; CELL, carboxyl ester
lipase like; FAPP, feto-acinar pancreatic protein.
Enzyme: bile salt-stimulated lipase (EC 3.1.1.3).
(Received 26 June 2001, revised 5 November 2001, accepted
8 November 2001)
Trang 2milk [27±29] Variants of higher, as well as lower, molecular
mass than the most common 120-kDa variant were
detected Occasionally, two different variants occurred
simultaneously in the same milk sample, e.g a BSSL
variant of the most commonly occurring molecular mass
coexisted with a variant of lower or higher mass The
differences in molecular masses were shown to reside in the
C-terminal part of the protein, but could not be explained
by differences in carbohydrate content Rather it was
speculated that it is the number of proline-rich repeats that
varies [28]
In the present study we show that a hypervariable region
located to exon 11 in the BSSL gene explain the different
forms of BSSL found in human milk Moreover, we show
that several molecular variants occur and that some 56% of
the Swedish population do have variants different from the
most common one of 16 repeats We speculate that this may
be of clinical signi®cance
E X P E R I M E N T A L P R O C E D U R E S
Collection of milk and blood samples
Human milk was collected via breast pump from healthy
women during their ®rst weeks of lactation The milk was
either used immediately (for RNA preparation) or stored at
)20 °C until analyzed Blood samples were collected in
vacutainerÒ tubes containing EDTA The samples were
stored at )70 °C until DNA was isolated
SDS/PAGE and Western blotting
One milliliter of human milk was centrifuged at 15 800 g for
10 min and the fat layer was discarded The skimmed milk
was diluted 10-fold, after which 10 lL was applied to a 10%
SDS/PAGE [30] After gel-electrophoresis, Western blotting
was performed using BSSL speci®c antibodies as previously
described [28]
BSSL cDNA [16], was used as template to create probe A and probe B Probe C was ampli®ed using a BSSL genomic clone, pS453 (L Hansson, Arexis AB, MoÈlndal, Sweden, personal communication) as template PCR was performed
in a total volume of 30 lL (50 ng plasmid DNA, 10 mM Tris/HCl, pH 8.3, 1.5 mMMgCl2,50 mMKCl, 2 lMeach of dCTP, dGTP, and dTTP, 0.82 lM[a-32P] dATP, 7.5 pmol
of each primer, and 2.5 U of Taq polymerase) The reactions were carried out for 30 cycles with denaturation at 94 °C for
30 s, annealing at 55 °C for 1 min and extension at 72 °C for
1 min The program ended with an elongation at 72 °C for 7 min The PCR products were puri®ed on a Sephadex G-50 ÔNick columnÕ (Amersham Pharmacia Biotech, Uppsala, Sweden) before used in hybridization experiments RNA isolation and Northern blot hybridization
Total RNA was isolated from fresh human milk samples as previously described [31] RNA hybridization was per-formed essentially as described in Sambrook et al [32] Approximately 20 lg of each RNA preparation was separated on 1% agarose gels, blotted onto Hybond-N
®lters (Amersham International plc., Buckinghamshire, UK) and hybridized to a [32P]dATP-labelled probe After hybridization, ®lters were washed and signals visualized using a Molecular Imager (Bio-Rad Laboratories, Hercules, CA).32P-Labelled k HindIII digested DNA was used as molecular mass standard on the RNA gels
DNA isolation and Southern blot hybridization Genomic DNA was isolated from 10 mL EDTA-blood as previously described [33] For Southern blot analysis, 10 lg
of DNA was digested with appropriate restriction enzyme(s) Digested DNA was separated on an agarose gel, transferred to a Hybond-N ®lter (Amersham) and hybridized to a [32P]dATP-labelled probe as described in Sambrook et al [32] Pre-hybridization, and hybridization, was performed at 42 °C in standard solutions,
supplemen-Table 1 Oligonucleotide primers used to amplify DNA probes Positions refer to the sequence of the BSSL gene submitted to the EMBL databank [22], accession no M94579.
Probe A
BSSL03 5¢-GACCCCAACATGGGCGACTC-3¢ 10621±10640 BSSL04 5¢-GTCACTGTGGGCAGCGCCAG-3¢ 10793±10774 Probe B
SYM2677 a 5¢-tctagaagcttGGCGCCGTGTACACAGAAGGTGGG-3¢ 4047±4069 SYM2133: 5¢-GTTGGCCCCATGGCCGGACCCCAT-3 4752±4729 Probe C
SYM2143 a 5¢-cgggatccGAAGCCCTTCGCCACCCCCACG-3¢ 10201±10222 BSSL05 5¢-GGCCTCGTGGTGGGAGGCCCTT-3¢ 10336±10357
a The ®rst 11 bases in primer SYM2677 and the ®rst eight bases in primer SYM2143 are linkers with no relevance for the application in this paper.
Trang 3ted with 50% formamide After washing the ®lter, signals
were visualized using Molecular Imager (Bio-Rad)
32P-Labelled k HindIII DNA was run in parallel as a size
marker
Cloning and DNA sequencing
The region of repeats in BSSL exon 11 was PCR ampli®ed
using the PlatinumÒ Pfx DNA polymerase (Life
Technol-ogies Inc., Gaithersburg, MD, USA) To improve
ampli®-cation of the extremely GC-rich repeats, betaine was added
to each reaction to a ®nal concentration of 2M (Sigma,
St Louis, MO, USA) A pair of primers, referred to as
BSSL 12 and BSSL 14, was designed to cover the entire
sequence of repeats (BSSL 12: 5¢-ACCAACTTCCT
GCGCTACTGGACCCTC-3¢; BSSL 14: 5¢-GGAGCC
CCTGGGGTCCCACTCTTGT-3¢) The PCR started with
a denaturation step (96 °C, 5 min) followed by 35 cycles
with denaturation (96 °C, 45 s) and annealing/elongation
(68 °C, 5 min) The reaction terminated by a ®nal
incuba-tion at 68 °C for 10 min
The PCR products were separated on an agarose gel and
the fragments to be cloned were recovered using Gene-clean
II (BIO 101, Carlsbad, CA, USA) Cloning was performed
using the pGEMÒ-T easy vector system II (Promega Co.,
Madison, WI, USA) Before ligation into the pGEMÒ-T
easy vector, the PCR fragments had to be modi®ed using the
A-tailing procedure for blunt-ended PCR fragments, as
recommended by Promega
The cloned fragments were sequenced on both strands
using the Big Dye terminator kit (PE Applied Biosystems,
Foster City, CA) supplemented with betaine to a ®nal
concentration of 1M (Sigma) BSSL 12 or BSSL 14
(described above) were used as primers, and the DNA
was ampli®ed for 30 cycles with denaturation at 98 °C (30 s)
and annealing/elongation at 60 °C (5 min) The reactions
were analyzed on an ABI PRISM 377A DNA sequencer
(PE Applied Biosystems)
R E S U L T S
Expression of different BSSL variants in human milk
To con®rm the described heterogeneity in molecular mass of
milk derived BSSL and select representatives for different
BSSL phenotypes we screened milk samples from nine
different mothers The milk proteins were separated on
SDS/PAGE, electroblotted and immunostained with BSSL
speci®c antibodies (Fig 1) The most commonly occurring variant of BSSL migrated with an apparent molecular mass
of 120 kDa (donors D11, D8, D7) A variant with an apparently lower molecular mass, i.e 100 kDa, was found in some milk samples, either as the only one (donor D2) or coexpressed with a variant of the most common molecular mass (donors D6 and D3) A single mother (donor D1) had a variant with higher molecular mass (160 kDa) than the most common one This mother also carried the 100 kDa variant in her milk Donors D4 and D5 carried only the 120-kDa BSSL variant in their milk samples (data not shown)
Analysis of BSSL transcripts in milk cells Northern blot hybridization was performed on RNA isolated from milk from four different mothers, D1 and D6±D8 (Fig 2) A 2.8-kb transcript was detected in RNA from mothers D7 and D8 when a fragment complementary
to a sequence immediately upstream the repeats in exon 11 was used as a probe (probe A; Fig 3) A slightly shorter transcript, 2.7 kb, was detected in RNA isolated from mother D6 The RNA isolated from mother D1 contained two hybridizing transcripts, 2.7 and 3.0 kb in size, respec-tively To exclude the possibility that probe A had failed to detect any possible truncated BSSL mRNA we used another probe, complementary to exon 2 to exon 4 in the BSSL cDNA (probe B; Fig 3) However, identical results
as with probe A were obtained using probe B in the Northern blot (data not shown)
Genetic variation occurs in exon 11 of theBSSL gene
To explore the possibility that genetic rearrangement(s) within the BSSL gene might explain the occurrence of
Fig 1 Naturally occurring variants of the BSSL protein in human milk.
Milk proteins from seven dierent donors (D6, D11, D1, D8, D3, D2
and D7) were separated on a 10% SDS/PAGE and immunostained
with BSSL speci®c antibodies The molecular mass standards are
shown on the right.
Fig 2 Northern blot analysis of total RNA from milk cells isolated from four dierent mothers (D1, D6, D7 and D8) The RNA was hybridized to a BSSL speci®c probe, Probe A HindIII-cut k was used
as the molecular mass marker (M).
Trang 4molecular mass variants of BSSL, we isolated DNA from
eight of the mothers (D1±D8) and performed Southern blot
hybridizations (Fig 4) PstI digested DNA was hybridized
to a probe complementary to a sequence in BSSL exon 11
(probe A; Fig 3) According to the published BSSL
sequence [7±10] this probe was expected to hybridize to a
731-bp PstI fragment carrying all 16 repeats Accordingly, a
0.7-kb PstI fragment was detected in all DNA samples
isolated from mothers carrying the most common variant of
BSSL in their milk, i.e D3±D8 However, this 0.7-kb PstI
fragment was not found in DNA from mother D2, carrying
only the low molecular mass variant in her milk Instead, D2
and also the other mothers carrying low molecular mass
variants in their milks (D1, D3 and D6) carried a shorter
PstI fragment (0.6 kb) The mother with a high molecular
mass BSSL variant in her milk (D1), carried a longer
hybridizing PstI fragment (0.9 kb) not detected in any other
DNA sample Also a third PstI fragment (0.7 kb) was
detected in DNA from mother D1 In contrast to the 0.9
and 0.6 kb fragments this 0.7-kb fragment did not correlate
with any BSSL protein variant in milk from mother D1
When the DNA samples were digested with EcoRI and
hybridized to probe C (Fig 3) the hybridizing fragments
corresponded to the products obtained with PstI digestion
and probe A (Fig 4b) DNA isolated from donors
expressing the most common BSSL variant in milk (D3±
D8) yielded a 2.2-kb EcoRI fragment when hybridized to
probe C A shorter fragment (2.1 kb) was detected in DNA
isolated from donors carrying the 100-kDa variant of BSSL
in milk (D1±D3 and D6) In DNA isolated from mother
D1, three EcoRI fragments were found to hybridize to
probe C (2.1, 2.2, and 2.4 kb, respectively)
Several other appropriate restriction enzymes and DNA
probes were used to cover the entire BSSL gene, looking for
additional genetic rearrangements However, no genetic
variation was detected in any other part of the BSSL gene,
neither upstream nor downstream the repeats in exon 11
(data not shown) Hence, we conclude that rearrangements
(deletions and insertions) occur within the region carrying
the repeats in exon 11 of the BSSL gene
PCR ampli®cation, cloning and DNA sequencing
of different BSSL alleles
To further characterize some of the rearrangements in BSSL
exon 11, we used PCR to amplify the region carrying the
repeats in DNA isolated from two mothers (D1 and D2)
(Fig 5) According to the published sequence [7±10] a
678-bp fragment was expected to amplify if all the 16 repeats
(33 bp each) is present and if there is no deletions or
insertions The results of the PCR con®rmed the Southern
blot results, i.e both mothers carry a deletion within one
(D1) or both (D2) alleles of their BSSL gene In addition, D1 also carries an insertion within another allele, shown by the ampli®cation of a fragment 0.9 kb in size Also in concert with data from Southern blot, a third fragment corresponding to the size of the wild-type allele (678 bp) was detected in DNA from D1
The 0.6-kb PCR fragments, expected to carry the proposed deletions, were cloned from each of the samples (D1 and D2) and the DNA sequenced When the sequences were aligned to the previously published DNA sequence, it was con®rmed that the deletions had occurred within the region of repeats (Fig 6) However, the deletions were not identical between the two samples The fragment that was sequenced from mother D1 was shown to carry a 98-bp deletion that changes the reading frame of the gene and predicts a premature translational stop after 632 amino acids (Fig 7) The sequence from mother D2 was essentially identical to D1 except that one basepair less was deleted, i.e a 97-bp deletion was found This difference predicts an even earlier translational stop, i.e after 610 amino acids In both cases the deletion changes the reading frame and predicts a new C-terminal tail (RAAHG) Besides the deletions, the sequences of D1 and D2 were identical to the published sequence except for one base substitution that does not affect the protein sequence (Fig 6)
TheBSSL gene contains a hypervariable region
in exon 11
To estimate the frequency of the BSSL polymorphism in a larger population, DNA was isolated from 295 healthy blood donors, digested with PstI and hybridized to probe
A (Fig 3) in Southern blot experiments A high frequency
of variation was found Only 131 out of the 295 (44%) DNA samples showed a restriction pattern corresponding
to the published sequence, i.e a PstI fragment 731 bp in size In 23 out of 295 (8%) analyzed DNA samples, a PstI fragment considerably shorter than 731 bp was detected
As many as 41% (121/295) of the analyzed DNA samples showed a heterozygous pattern with one PstI fragment
731 bp in size and another fragment considerably shorter An increased length of the actual PstI fragment was found in 21 out of 295 (7%) of the analyzed DNA samples
D I S C U S S I O N
The BSSL locus is known to exhibit a high degree of polymorphism [23], but whether this polymorphism affects the BSSL coding region has not previously been shown Therefore, in the present paper we have investigated if the
Fig 3 Schematic drawing of the genetic organization of the human BSSL gene, modi®ed from Lidberg et al [22] Exons are shown as boxes and numbered 1±11 The repeated region in exon 11 (rep) is hatched Horizontal bars show the position of sequence homology to probes A, B and C, used for hybridization experiments Cleavage sites for PstI (P) and EcoRI (E) are marked.
Trang 5occurrence of different BSSL variants in human milk is due
to genetic variation within the BSSL gene
We collected milk samples and isolated RNA and DNA
from nine lactating mothers Southern blot hybridization
experiments con®rmed the occurrence of allelic variance in
the BSSL gene The variations were exclusively found
within a PstI fragment covering a region of direct repeats in
exon 11, and in each woman a correlation to the molecular
mass of BSSL in milk was evident Mothers known to have
low molecular mass variant(s) of the BSSL protein in their
milk were shown to carry a deletion, 0.1 kb in size, within
this PstI fragment Mothers with two different BSSL
variants in their milk, e.g the most common 120-kDa variant together with one of lower mass, carried the deletion
in one of the alleles, whereas the mother with only the low molecular mass variant in milk (D2) carried deletions in both alleles In DNA isolated from mother D1, known to express a high molecular mass variant (160 kDa) together with a low molecular mass variant in her milk, PstI fragments of 0.6 and 0.9 kb were detected The sizes of these fragments correspond to a 0.1-kb deletion in one allele, and
a 0.2-kb insertion in another allele, and are likely to encode the low and high molecular mass variants detected in milk from D1, respectively However, a third, unexpected PstI
Fig 4 Southern blot hybridization (A) DNA
isolated from donors D1±D8 was digested
with PstI and hybridized to probe A (Table 1,
Fig 3) Hybridizing fragments shorter than
0.56 kb correspond to fragments within the
pseudogene CELL HindIII-cut k was used
as molecular mass marker (M) (B) DNA
isolated from the same donors as above was
digested with EcoRI and hybridized to probe
C (Table 1, Fig 3) HindIII-cut k was used
as molecular mass marker (M).
Trang 6of the protein were found in milk from this donor.
A DNA fragment covering the deletions in exon 11 was PCR ampli®ed, cloned and sequenced from two different mothers (D1 and D2) The DNA sequences con®rmed the deletions and the deduced amino-acid sequences predicted BSSL variants of considerably lower molecular mass, i.e the variants were predicted to be truncated after 632 and 610 amino acids, respectively Hence, these BSSL variants are truncated within the region of proline rich repeats and the number of repeats is decreased from 16 to 8.5 and 6.5, respectively, in the D1 and D2 variants In both variants, a new C-terminal sequence consisting of ®ve amino acids (RAAHG) is created due to the deletions In the wild-type protein (the most common variant), the C-terminal consists
Fig 6 The DNA sequence of the repeated region carrying a deletion in exon 11 from mother D1 and D2 was aligned to the published BSSL sequence (wt) The repeats are numbered 1±16 according to the wt sequence Alignments were performed using the program BESTFIT from the University of Wisconsin GCG software package Dots represent gaps that were inserted to improve alignment The position of primers 12 and 14 used for ampli®cation and sequencing of the fragments are marked An asterisk (*) marks the position of the single base substitution detected in D1 and D2.
Fig 5 PCR analysis of BSSL exon 11 DNA from mother D1 and D2
was ampli®ed using a pair of primers covering the entire region of
repeats in the BSSL gene Three independent reactions were run from
each mother Lane 1±3, D1; lane 4±6, D2 The shortest fragments,
0.6 kb, were subsequently cloned and sequenced.
Trang 7of the 16 repeats followed by a hydrophobic tail of 11 amino
acids The low molecular mass variants expressed by
mothers D1 and D2 did not react with speci®c antibodies
directed towards this tail (M StroÈmqvist, AstraZeneca
R & D, MoÈlndal, Sweden, personal communication)
con-®rming a different sequence of the tail The function of the
tail has previously been discussed in the literature Deletion
of the tail by in vitro mutagenesis of the human enzyme was
shown to signi®cantly decrease expression of the protein,
presumably by affecting mRNA stability [16] From studies
on the crystal structure of bovine BSSL it was concluded
that the terminal six hydrophobic amino acids physically
block a putative oxyanion hole at the active site
Calcula-tions indicated that removal of this hexapeptide exposes a
large hydrophobic area on the protein surface suggesting
that displacement of this region can play a role in the
stability and function of BSSL [34]
The size of the BSSL transcript has previously been
estimated to be 2.5 or 2.9 kb [7,9] We detected BSSL
transcripts of 2.8 kb in Northern blots performed on RNA
from two mothers (D7 and D8) known to have the most
common BSSL genotype and phenotype in milk RNA
isolated from mother D1, expressing the high molecular
mass variant together with a low molecular mass variant of
BSSL in milk, carried two transcripts that hybridized to the
BSSL-speci®c probe The sizes of these two transcripts were
estimated to be 2.7 and 3.0 kb, respectively Accordingly, we
expected to ®nd two transcripts in RNA from mother D6,
known to have two BSSL variants (100 + 120 kDa) in
milk, and to carry the exon 11 deletion in one of the BSSL
alleles However, only one transcript was detected The
band representing this transcript is however broad and we
believe that the resolution of the gel was insuf®cient to
separate the two proposed transcripts
The frequency of variation in exon 11 of the BSSL gene
was determined by Southern blot experiments with DNA
isolated from 295 blood donors When compared to the
published sequence [7±10] 56% of the individuals examined
carried genetic variations within the repeats These data
con®rm that BSSL is located in a hypervariable region [23]
but also shows that the polymorphism is due to deletions or
insertions within the BSSL coding sequence Hence, we
conclude that exon 11 in the BSSL gene consists of a
hypervariable region and that the current understanding
that exon 11 of the human gene encodes 16 proline-rich
repeats is an oversimpli®cation and needs to be revisited
This high frequency of variation in the BSSL gene corresponds very well with a previous study on incidence
of molecular forms of BSSL in human milk [29] This study showed that 50% of the milk samples contained BSSL variants with a molecular mass different to the most common variant
An onco-fetal variant of BSSL, denoted feto-acinar pancreatic protein (FAPP), has been detected in human embryonic and fetal pancreas and in pancreatic tumoral cell lines [35,36] FAPP and BSSL are structurally closely related, but are distinguished by a monoclonal antibody directed towards a fucosylated epitope, present on FAPP but not on BSSL [37] Compared to BSSL, FAPP has lower enzymatic activity against ester substrates, and is poorly secreted [36,37] The cDNA sequence of FAPP is identical to that of BSSL except for a 330-bp deletion in the C-terminal repeated region [38,39] The fact that we now show that 50% of a Swedish population carry a deletion in the repeated region of the BSSL gene makes it tempting to speculate that FAPP is identical to a naturally occurring low molecular mass variant of BSSL The characteristic FAPP epitope should then result from tissue speci®c glycosylation, rather than structural features of the protein If so, the concept of FAPP being an onco-fetal variant of BSSL, exclusively expressed in proliferating cells such as embryonic and fetal pancreas as well as pancreatic tumoral cells, should be re-evaluated The human hepa-toma cell line HepG2 also expresses a BSSL isoform of lower molecular mass [40] The cDNA sequence of this isoform contained only one ÔrepeatÕ
The obvious question is, of course, whether there are biological phenotypes associated with speci®c BSSL vari-ants? As mentioned above, it has been proposed that there is
no signi®cant difference in enzymatic activity, bile salt stimulation, pH stability and temperature stability between BSSL of the most common molecular mass and variants of lower or higher mass [27,28] However, some low molecular mass variants with only half the speci®c activity compared
to the most common variant have been isolated and the concentration of BSSL was considerably lower in milk from mothers carrying only low molecular mass variant(s) [28]
A possible explanation of these somewhat contradictory results could be the presence or absence of the most C-terminal 11 amino acids, referred to as the tail Two low molecular mass variants characterized in this paper were both shown to lack the ÔnormalÕ C-terminal tail, whereas StroÈmqvist et al [28] showed that the tail is present in other low molecular mass variants From the crystal structure of bovine BSSL the tail was suggested to be involved in the active site machinery [34]
Finally, a positive correlation has been demonstrated between BSSL activity in serum, assayed as cholesterol esterase activity, and serum cholesterol levels [5,6] More-over, in vitro BSSL was shown to transform larger LDL particles to smaller, more atherogenic LDL particles [41] Considering the data presented in the present paper, it is interesting to note that an association between BSSL genotype and serum lipid levels has been suggested [24,25] Taken together, we have shown that the molecular mass variants of BSSL found in milk results from a polymor-phism in the BSSL gene This strongly suggests that BSSL variants described in other tissues, such as the onco-fetal protein FAPP, is due to the same frequently occurring
Fig 7 Comparison of the deduced amino-acid sequences of the repeats
in BSSL from the published sequence carrying 16 repeats (wt), and the
shorter variants from mother D1 and D2.
Trang 8We are grateful to Yvonne Andersson for excellent technical assistance
and to Mats StroÈmqvist for fruitful discussions Grants from the
Swedish Medical Research Council (05708 and 12721), Astra-HaÈssle
AB, PPL therapeutics, Margarinindustrin, Stiftelsen Oskarfonden,
VaÈsterbotten County Council, and The Swedish Society for Medical
Research (postdoctoral fellowship to S L.) supported this work.
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