Báo cáo Y học: Human bile salt-stimulated lipase has a high frequency of size variation due to a hypervariable region in exon 11 pot

Human bile salt-stimulated lipase has a high frequency of size variation due to a hypervariable region in exon 11 Susanne Lindquist 1 , Lars BlaÈ ckberg 2 and Olle Hernell 1 Departments of 1 Clinical Sciences, Pediatrics and 2 Medical Biosciences, Medical Biochemistry, Umea Ê University, Sweden The a pparent 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 dierences to reside in a region o f 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 e xplanation for t he molecular v ariants of BSSL found in milk. By Southern blot hybridization we analyzed the BSSL gene from mothers known t o have BSSL of dierent molecular masses in their milk. A polymorphism was found within exon 11, previously shown t o c onsist o f 1 6 n ear i dentical repeats of 3 3 bp each. We detected deletions or, in one case, a n insertion corres- ponding to the variation in molecular mass of the BSSL protein found in milk from the respective woman. Fur- thermore, we f ound 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 a nd 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; r epeats; po ly- 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 a nd contributes to the hydrolysis of dietary mono-, di-, a nd 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 i n s erum [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 b een reported t o consist of a unique structure with 16 proline-rich, O-glycosylated repeats of 11 amino- acid residues each [7±10]. T he 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 t he 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 t he 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 i s well c onserved between species, but the number of proline-rich repeats varies, from three in cow and mouse [19,20] t o 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 p olymorphism [23]. A co rrelation 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 a nd 9, whereas there are some major differences in other parts. A 4.8-kb fragment, spanning exons 2±7 in the BSSL ge ne, i s deleted in CELL .Thereare also several base substitutions within exons 10 and 11. A region in exon 11, encoding the proline-rich repeats, differs between BSSL and CELL.HumanBSSL has previously been shown to carry 16 repeats, although in t his Correspondence to S. Lindquist, Department of Clinical Sci ences, Pediatrics, Umea Ê University, SE-901 85 Umea Ê ,Sweden. Fax: + 4 6 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: b ile salt-stimulated lipase (EC 3.1.1.3). (Received 2 6 June 2 001, revised 5 November 200 1, accepted 8 November 2001) Eur. J. Biochem. 269, 759±767 (2002) Ó FEBS 2002 paper we show that the number of r epeats can vary between individuals. In CELL, this region has been described as a hypervariable region and the o verall number o f repeats are fewer compared to BSSL. Naturally occurring variants of BSSL, differing in apparent molecular mass, have been described in human milk [27±29]. V ariants of h igher, as well as lower, molecular mass than the most common 120-kDa variant were detected. Occasionally, two different variants occurred simultaneously in the s ame m ilk sample, e .g. a BSSL variant of the most commonly occurring molecular mass coexisted with a variant o f l ower or higher mass. The differences in molecular masses w ere s hown to re side i n t he 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 t he p resent study we show that a hypervariable region locatedtoexon11intheBSSL gene explain t he different forms of BSSL found in human milk. Moreover, we show that several m olecular variants occur and that some 56% of the S wedish population do have variants different from the most common one of 16 repeats. We speculate that this may be of clinical signi®cance. EXPERIMENTAL PROCEDURES 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 h uman milk was centrifuged at 15 800 g for 10 min a nd the f at layer w as discarded. The s kimmed milk was diluted 10-fold, after which 10 lLwasappliedtoa10% SDS/PAGE [30]. After gel-electrophoresis, Western blotting was performed using B SSL speci®c antibodies as pr eviously described [28]. Probes for hybridization experiments DNA fragments to be used as probes in Northern or Southern blot hybridizations were obtained by PCR ampli- ®cation. The primers used for a mpli®cation of each probe are list ed in Table 1. Plasm id pS146, c arrying the entire BSSL cDNA [16], was used as template to create probe A and probe B. Probe C was ampli®ed using a B SSL genomic clone, pS453 (L. Hansson, Arexis AB, Mo È lndal, Sweden, personal c ommunication) as template. PCR was performed in a total volume of 30 lL (50 ng plasmid DNA, 10 m M Tris/HCl, p H 8.3, 1.5 m M MgCl 2, 50 m M KCl, 2 l M each of dCTP, dGTP, and dTTP, 0 .82 l M [a- 32 P] dATP, 7.5 pmol of each primer, and 2.5 U of Ta q polymerase). The reactions were carried out for 30 cycles with d enaturation at 9 4 °Cfor 30 s, annealing at 55 °C for 1 min a nd extension at 72 °Cfor 1 min. The program ended with a n elongation a t 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 experime nts. 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 [ 32 P]dATP-labelled probe. After hybridization, ®lters were washed and signals visualized using a Molecular I mager (Bio-Rad L aboratories, Hercules, CA). 32 P-Labelled k HindIII digested DNA was used as molecular mass standard on the R NA gels. DNA isolation and Southern blot hybridization Genomic DNA was isolated from 10 m L 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 [ 32 P]dATP-labelled probe as described in Sambrook et al . [32]. Pre-hybridization, and hybridization, was performed at 42 °C in s tandard solutions, supplemen- Table 1. Oligonucleotide primers used t o a mplify D NA probes. Position s refer to the sequence of the BSSL gene subm itted to the EMB L databank [22], a ccession no. M94579. Probe Primer sequence Positions 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. 760 S. Lindquist et al. (Eur. J. Biochem. 269) Ó FEBS 2002 ted with 50% formamide. After washing the ®lter, signals were visualized using Molecular Imager (Bio-Rad). 32 P-Labelled k HindIII DNA was r un in parallel as a size marker. Cloning and DNA sequencing The region o f r epeats in BSSL exon 11 was PCR ampli®e d using t he Platinum Ò Pfx DNA polymerase (Life Technol- ogies Inc., Gaithersburg, MD, USA). To improve ampli®- cation of the extremely GC-rich repeats, betaine was a dded to each reaction to a ®nal concentration of 2 M (Sigma, St Louis, MO, U SA). A pair of primers, referred to as BSSL 12 and BSSL 14, was designed to cover the entire sequence of repeats (BSSL 12: 5¢-ACCAAC TTCCT GCGCTACTGGACCCTC-3¢;BSSL14:5¢-GGAGCC CCTGGGGTCCCACTCTTGT-3¢). The P CR started with adenaturationstep(96°C, 5 min) followed by 35 cycles with denaturation (96 °C, 45 s) and annealing/elongation (68 °C, 5 min). The reaction terminated b y a ®nal i ncuba- tion at 68 °C for 10 min. The PCR p roducts were sep arated on an agarose gel and the fragments to be cloned were recovered using Gene-clean II (BIO 101, Carlsbad, CA, USA). Cloning was p erformed using t he pGEM Ò-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 p rocedure for blunt-ended PCR fragments, as recommended by Promega. The cloned fragments were sequenced on both strands using t he Big Dye terminator kit (PE Applied Biosystems, Foster City, CA) supplemented with betaine to a ®nal concentration of 1 M (Sigma). BSSL 12 or BSSL 14 (described above) were used as primers, and t he DNA was a mpli®ed for 30 cycles with denaturation a t 9 8 °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). RESULTS Expression of different BSSL variants in human milk To con®rm the described heterogeneity in m olecular m ass o f milk derived BSSL and select representatives for different BSSL phenotypes we screened milk samples from nine different mothers. The m ilk proteins were separated on SDS/PAGE, electroblotted and immunostained with BSSL speci®c antibodies (Fig. 1). The most c ommonly occurring variant of BSSL migrated with an appar ent molecular mass of  120 kDa (donors D11, D8, D7). A variant with an apparently lower m olecular mass, i.e.  100 kDa, was found in some milk samples, either as the only on e (donor D2) or coexpressed with a variant of the most common molecular m ass (donors D6 and D3). A single mother (donor D1) had a varian t with higher molecular m ass (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 m ilk from four different mothers, D1 and D6±D8 ( Fig. 2). A 2.8-kb transcript was detected in RNA from mother s D7 a nd D8 when a fragment c omplementary to a sequence immediately upstream t he repeats i n exon 1 1 was used as a probe (probe A; Fig. 3). A slightly shorter transcript,  2.7 kb, was d etected 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 e xc lude the possibility that probe A had failed t o 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 w ere obtained using probe B in t he Northern blot (data not shown). Genetic variation occurs in exon 11 of the BSSL 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 dierent dono rs (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 shownontheright. Fig. 2. Northern blot analysis of total RNA from milk cells isolated from four dierent mothers (D1, D6, D7 and D8). The RNA was hybridized to a BSSL speci®c probe, P robe A. HindIII-cut k was u sed as the m olecular mass m arker (M). Ó FEBS 2002 Molecular mass variants of BSSL in human milk (Eur. J. Biochem. 269) 761 molecular mass variants of B SSL, we isolated DNA from eight of the mothers (D1±D8) and p erformed Southern blot hybridizations (Fig. 4). PstI digested DNA was hybridized to a p robe 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 c arrying the most common variant of BSSL in their m ilk, i.e. D3±D8. However, this 0.7-kb PstI fragment was not found in DNA from m other D2, carrying only the low molecular mass variant in her m ilk. I nstead, D 2 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 s ample. 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 t his 0.7-kb f ragment did not correlate with any BSSL protein variant in milk from mother D1. When the DNA samples were digested with Eco RI and hybridized to probe C (Fig. 3) the hybridizing fragments corresponded t o the products obtained with PstIdigestion 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 c arrying the 100-kDa variant of BSSL in milk (D1±D3 and D6). In DNA isolated from mother D1, thre e EcoRI fragments were foun d 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 t he published sequence [7±10] a 678-bp fragment was expected to amplify if all the 1 6 r epeats (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 a nother allele, shown by the ampli®cation of a fragment  0.9kbinsize.Alsoin concert w ith d ata f rom Southern blot, a third fragme nt corresponding to the size of the wild-type a llele (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 t he samples (D1 and D2) and the DNA sequenced. When the sequences were aligned to the previously published DNA s equence, it was con®rmed that t he deletions had occurred within th e region of repeats (Fig. 6). However, the deletions were not identical between the two samples. The f ragment t hat 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 f rom 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 c ases t he d eletion changes the reading frame and predicts a new C-terminal tail (RAAHG). Besides the d eletions, t he seq uences of D1 and D 2 w ere i dentical to the published sequence e xcept for one base substitution that does not affect the protein sequence (Fig. 6). The BSSL gene contains a hypervariable region in exon 11 To estimate the frequency of the BSSL polymorphism in a larger population, DNA was isolated from 2 95 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) o f 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. DISCUSSION The BSSL locus is known t o exhibit a high degree o f 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, m odi®ed f rom Lidberg et al .[22].Exons are shown as boxes and numbered 1±11. The r epeated reg ion in exon 1 1 (re p) is hatch ed. Horiz ontal bars show the p osition o f sequ ence homology t o probes A , B and C , used for hybridization experiments. C leavage sites f or PstI(P)andEcoRI (E) are marked. 762 S. Lindquist et al. (Eur. J. Biochem. 269) Ó FEBS 2002 occurrence of different BSSL variants in human milk is du e to genetic variation with in 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 Pst I fragment covering a region of direct repeats i n 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 i n their milk were shown to carry a deletion,  0.1 kbinsize,within this PstI fragment. Mothers with two differen t BSSL variants in t heir milk, e.g. the most common 120-kDa variant together with o ne of l ower mass, carried the deletion in one of the alleles, whereas t he mother with only the low molecular mass variant in milk (D2) carried deletions in both alleles. In DNA isolated from mother D1, known t o express a high molecular m ass variant (160 kDa) together with a low molecular mass variant in her m ilk, PstI fragments of 0 .6 and 0 .9 kb were detected. The sizes of these fragments c orrespond to a 0.1-kb deletion i n one allele, and a 0 .2-kb i nsertion in another a llele, 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 d ono rs D1±D8 w as digested with PstI a nd h ybrid ized to probe A (T ab le 1, Fig. 3). Hybridizing fragments shorter t han 0.56 kb correspond t o fragments within the pseudogene CELL. HindIII-cut k was used as molecular m ass marker (M). (B) DN A isolated from th e same donors a s above was digested with EcoRIandhybridizedtoprobe C(Table1,Fig.3).HindIII-cut k was used as molecular m ass marker (M). Ó FEBS 2002 Molecular mass variants of BSSL in human milk (Eur. J. Biochem. 269) 763 fragment, was detected in DNA from mother D1. This fragment was  0.7 kb, corresponding to a fragment size probably carrying the most commonly occurring 16 repeats. This fragment probably originates from a duplication of the BSSL gene, or at least of the 5¢ end, in one of the alleles. This duplicated copy was not expressed as on ly two variants of the protein were found in milk from this donor. A DNA fr agment 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 t he deletions and the deduced amino-acid sequences predicted BSSL variants of considerably lower molecular m ass, 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 r ich repeats and the number o f repeats is decreased from 16 t o 8.5 and 6.5, respectively, in the D1 and D2 variants. In both variants, a new C-terminal sequence consisting of ®ve a mino acids (RAAHG) is created due to the delet ions. In the wild-type protein (the most common v ariant), the C -terminal consists Fig. 6. The DNA sequence of the rep eated region c arrying a de letion in exon 11 from mother D1 and D2 w as aligned t o the published BSSL sequence (w t). The repeats are numbered 1±16 according to the wt sequence. Alignments were performed using the program BESTFIT from the U niversity of Wisconsin GCG software packa ge. Dots represent gaps that were inserted to improve alignment. The position of primers 12 and 14 used f or ampli®cation and sequencing of the fragments a re marked. A n asterisk ( *) marks the position of the single base subs titution detected in D1 an d D2. Fig. 5. PCR analysis of BSSL exon 11. DNA f rom mother D1 and D2 was ampli®ed using a pair of primers covering the entire region of repeats i n t he BSSL ge ne. Three independent reactions were run from each mother. Lane 1±3, D1; lane 4±6, D2. The shortest fragments,  0.6 kb, we re subsequently cloned and seque nced. 764 S. Lindquist et al. (Eur. J. Biochem. 269) Ó FEBS 2002 of the 16 repeats followed by a hydrophobic tail of 11 amino acids. The low molecular mass variants expressed by mothers D1 and D2 d id not react with speci®c antibodies directed towards t his tail ( M. Stro È mqvist, AstraZeneca R&D,Mo È lndal, Sweden, personal communication) con- ®rming a different sequence of t he tail. The f unction 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 s tability [16]. F rom 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 t he active site. Calcula- tions indicated that removal of this hexapeptid e exposes a large hydrophobic area on the protein surface suggesting that displacement of this re gion can play a role in the stability and function of BSSL [34]. The s ize o f the BSSL transcript has p reviously been estimated to be  2.5 or 2.9 kb [7,9]. We detected BSSL transcripts of 2.8 kb in Northern blots p erformed on RNA from two mothers (D7 and D8) known to have the most common BSSL genotype and phenotype i n milk. RNA isolated fr om mother D1, expressing t he high molecular mass varian t together with a low molecular m ass v ariant of BSSL in milk, carried two transcripts that hybridized to the BSSL-speci®c probe. The sizes of these two transcripts were estimated t o b e 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 k Da) i n milk, and to carry t he 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 t o separate the two proposed transcripts. The frequency of variation in exon 11 of the BSSL gen e was determined by Southern blot experiments with DNA isolated from 295 blood donors. When compared to the published s equence [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 t hat the polymorphism is due to deletions or insertions within the BSSL coding sequence. Hence, we conclude that exon 11 in th e BSSL gene consists of a hypervariable r egion 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 m ass different to the m ost common variant. An onco-fetal variant of BSSL, d enoted feto-acinar pancreatic p rotein (FAPP), has been d etected 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 h as 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]. T he fact that we now show that  50% of a Swedish population c arry a deletion in the r epeated region of the BSSL gene makes i t 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, th e concept of F APP being an onco-fetal variant of BSSL, exclusively expressed in proliferating cells such as embryonic and fetal panc reas as well a s pancreatic tumoral cells, should b e r e-evaluated. The human hepa- toma cell line HepG2 also expresses a BSSL isoform of lower molecular m ass [ 40]. The cDNA sequence of t his 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 a nd temperature stability b etween BSSL of the most common molecular mass and variants of lower or higher m ass [27,28]. However, some l ow 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 co nsiderably lower in milk from mothers carrying only low molecular mass variant(s) [28]. A possible explanation of th ese somewhat contradictory results could be the presen ce o r absence of t he most C-terminal 11 amino acids, re ferred to as t he tail. Two l ow molecular mass variants characterized in this paper were bothshowntolacktheÔnormalÕ C-terminal tail, whereas Stro È mqvist et al. [ 28] showed that the tail is p resent 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 b een 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 s ame frequently occurring Fig. 7. Comparison of the deduced amin o-acid sequences of the repeats in B SSL from the published sequence carrying 16 repeats (w t), and the shorter v ariants from mother D1 and D2. Ó FEBS 2002 Molecular mass variants of BSSL in human milk (Eur. J. Biochem. 269) 765 genetic variation. Whether this polymorphism is correlated to activity of the enzyme a nd to serum lipid levels is currently under investigation. ACKNOWLEDGEMENTS We are grateful to Yvonne Andersson for excellent technical assistance andtoMatsStro È mqvist for fruitful d iscussions. Grants from t he Swedish Medical Research Council (05708 and 12721), Astra-Ha È ssle AB, PPL therapeutics, Margarinindustrin, Stiftelsen Oskarfonden, Va È sterbotten County Council, and The Swedish Society for Medical Research (postdoctoral f ellowship to S . L .) supported this w ork. 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