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

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Human bile salt-stimulated lipase has a high frequency of sizevariation due to a hypervariable region in exon 11Susanne Lindquist1, Lars BlaÈ ckberg2and Olle Hernell1Departments of1Clinical Sciences, Pediatrics and2Medical Biosciences, Medical Biochemistry, UmeaÊUniversity, SwedenThe a pparent molecular mass of human milk bile salt-stimulated lipase (BSSL) varies between mothers. Themolecular basis for this is unknown, but indirect evidencehas suggested the dierences to reside in a region o frepeats located in the C-terminal part of the protein. Wehere report that a polymorphism within exon 11 of theBSSL gene is the e xplanation for t he molecular v ariants ofBSSL found in milk. By Southern blot hybridization weanalyzed the BSSL gene from mothers known t o haveBSSL of dierent molecular masses in their milk.A polymorphism was found within exon 11, previouslyshown 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 BSSLprotein found in milk from the respective woman. Fur-thermore, we f ound that 56%, out of 295 individualsstudied, carry deletions or insertions within exon 11 in oneor both alleles of the BSSL gene. Hence, this is a hyper-variable region a nd the current understanding that exon 11in the human BSSL gene encodes 16 repeats is an over-simpli®cation and needs to be revisited. Natural variationin 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 lipaseis a digestive enzyme secreted from exocrine pancreas inall species examined. BSSL has a broad substratespeci®city a nd contributes to the hydrolysis of dietarymono-, di-, a nd tri-acylglycerols and is responsible fordigestion of fat-soluble vitamin esters and cholesterolesters in the small intestine. In some species, includinghumans, the gene is also expressed in the lactatingmammary gland and the resulting protein is a constituentof the milk [1,2]. Milk BSSL is a major reason whybreast-fed infants digest and absorb fat more ef®cientlythan formula-fed infants [3]. Moreover, BSSL has beendetected in low, but signi®cant, levels i n s erum [4]. Thefunction of BSSL in serum is unknown, but it has beensuggested to in¯uence the level of serum cholesterol [5,6].Deduced from the cDNA sequence, the human BSSLprotein consists of 722 amino acids with a predictedmolecular mass of 76 kDa [7±10]. The protein is, however,abundantly glycosylated and the apparent molecular masson SDS/PAGE has been estimated to 120±140 kDa[11,12]. Human BSSL has a unique primary structure ascompared to other mammalian lipases. The N-terminalpart of the protein shows striking homology to acetylcho-linesterase and some other esterases [7]. The C-terminalpart has b een reported t o consist of a unique structurewith 16 proline-rich, O-glycosylated repeats of 11 amino-acid residues each [7±10]. T he biological function of therepeated region is not fully understood. It has been shownthat the repeats protect the protein from denaturation byacid and from proteolysis by pepsin or pancreatic prote-ases in vitro [13,14]. It has also been shown that t heO-glycosylation of the repeated sequences is important forsecretion of rat pancreatic BSSL [15]. On the other hand,we and others have shown that t he repeats are completelydispensable for the typical functional properties of BSSL,i.e. catalytic activity, bile-salt activation, heparin binding,heat stability, stability at low pH and resistance toproteolytic inactivation [16±18].The BSSL protein i s well c onserved between species, butthe number of proline-rich repeats varies, from three incow and mouse [19,20] t o 16 in the human [7±10]. Thesalmon enzyme seems to be completely devoid of repeats[21].The human gene encoding BSSL spans 9.8 kb andconsists of 11 exons [22]. The gene has been mapped tochromosome 9q34- qter and the BSSL locus was shown toexhibit a high degree of p olymorphism [23]. A co rrelationbetween BSSL genotype and serum cholesterol levels hasbeen proposed [24,25] but to our knowledge, the polymor-phism in the BSSL gene has not been further characterizeduntil now.The carboxyl ester lipase like (CELL) gene is a ubiqui-tously transcribed pseudogene for BSSL [22,26]. Thesequence of the CELL gene is in some parts identical toBSSL, i.e. exons 1, 8 a nd 9, whereas there are some majordifferences in other parts. A 4.8-kb fragment, spanningexons 2±7 in the BSSL ge ne, i s deleted in CELL .Therearealso several base substitutions within exons 10 and 11.A region in exon 11, encoding the proline-rich repeats,differs between BSSL and CELL.HumanBSSL haspreviously been shown to carry 16 repeats, although in t hisCorrespondence to S. Lindquist, Department of Clinical Sci ences,Pediatrics, UmeaÊUniversity, SE-901 85 UmeaÊ,Sweden.Fax: + 4 6 90 123728, Tel.: + 46 90 7852128,E-mail: susanne.lindquist@pediatri.umu.seAbbreviations: BSSL, bile salt-stimulated lipase; CELL, carboxyl esterlipase 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, accepted8 November 2001)Eur. J. Biochem. 269, 759±767 (2002) Ó FEBS 2002paper we show that the number of r epeats can vary betweenindividuals. In CELL, this region has been described as ahypervariable region and the o verall number o f repeats arefewer compared to BSSL.Naturally occurring variants of BSSL, differing inapparent molecular mass, have been described in humanmilk [27±29]. V ariants of h igher, as well as lower, molecularmass than the most common 120-kDa variant weredetected. Occasionally, two different variants occurredsimultaneously in the s ame m ilk sample, e .g. a BSSLvariant of the most commonly occurring molecular masscoexisted with a variant o f l ower or higher mass. Thedifferences in molecular masses w ere s hown to re side i n t heC-terminal part of the protein, but could not be explainedby differences in carbohydrate content. Rather it wasspeculated that it is the number of proline-rich repeats thatvaries [28].In t he p resent study we show that a hypervariable regionlocatedtoexon11intheBSSL gene explain t he differentforms of BSSL found in human milk. Moreover, we showthat several m olecular variants occur and that some 56% ofthe S wedish population do have variants different from themost common one of 16 repeats. We speculate that this maybe of clinical signi®cance.EXPERIMENTAL PROCEDURESCollection of milk and blood samplesHuman milk was collected via breast pump from healthywomen during their ®rst weeks of lactation. The milk waseither used immediately (for RNA preparation) or stored at)20 °C until analyzed. Blood samples were collected invacutainerÒ tubes containing EDTA. The samples werestored at )70 °C until DNA was isolated.SDS/PAGE and Western blottingOne milliliter of h uman milk was centrifuged at 15 800 g for10 min a nd the f at layer w as discarded. The s kimmed milkwas diluted 10-fold, after which 10 lLwasappliedtoa10%SDS/PAGE [30]. After gel-electrophoresis, Western blottingwas performed using B SSL speci®c antibodies as pr eviouslydescribed [28].Probes for hybridization experimentsDNA fragments to be used as probes in Northern orSouthern blot hybridizations were obtained by PCR ampli-®cation. The primers used for a mpli®cation of each probeare list ed in Table 1. Plasm id pS146, c arrying the entireBSSL cDNA [16], was used as template to create probe Aand probe B. Probe C was ampli®ed using a B SSL genomicclone, pS453 (L. Hansson, Arexis AB, MoÈlndal, Sweden,personal c ommunication) as template. PCR was performedin a total volume of 30 lL (50 ng plasmid DNA, 10 mMTris/HCl, p H 8.3, 1.5 mMMgCl2,50 mMKCl, 2 lMeach ofdCTP, dGTP, and dTTP, 0 .82 lM[a-32P] dATP, 7.5 pmolof each primer, and 2.5 U of Ta q polymerase). The reactionswere carried out for 30 cycles with d enaturation at 9 4 °Cfor30 s, annealing at 55 °C for 1 min a nd extension at 72 °Cfor1 min. The program ended with a n elongation a t 72 °Cfor 7 min. The PCR products were puri®ed on a SephadexG-50 ÔNick columnÕ (Amersham Pharmacia Biotech,Uppsala, Sweden) before used in hybridization experime nts.RNA isolation and Northern blot hybridizationTotal RNA was isolated from fresh human milk samples aspreviously described [31]. RNA hybridization was per-formed essentially as described in Sambrook et al.[32].Approximately 20 lg of each RNA preparation wasseparated on 1% agarose gels, blotted onto Hybond-N®lters (Amersham International plc., Buckinghamshire,UK) and hybridized to a [32P]dATP-labelled probe. Afterhybridization, ®lters were washed and signals visualizedusing a Molecular I mager (Bio-Rad L aboratories, Hercules,CA).32P-Labelled k HindIII digested DNA was used asmolecular mass standard on the R NA gels.DNA isolation and Southern blot hybridizationGenomic DNA was isolated from 10 m L EDTA-blood aspreviously described [33]. For Southern blot analysis, 10 lgof DNA was digested with appropriate restrictionenzyme(s). Digested DNA was separated on an agarosegel, transferred to a Hybond-N ®lter (Amersham) andhybridized to a [32P]dATP-labelled probe as described inSambrook 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 PositionsProbe ABSSL03 5¢-GACCCCAACATGGGCGACTC-3¢ 10621±10640BSSL04 5¢-GTCACTGTGGGCAGCGCCAG-3¢ 10793±10774Probe BSYM2677a5¢-tctagaagcttGGCGCCGTGTACACAGAAGGTGGG-3¢ 4047±4069SYM2133: 5¢-GTTGGCCCCATGGCCGGACCCCAT-3 4752±4729Probe CSYM2143a5¢-cgggatccGAAGCCCTTCGCCACCCCCACG-3¢ 10201±10222BSSL05 5¢-GGCCTCGTGGTGGGAGGCCCTT-3¢ 10336±10357aThe ®rst 11 bases in primer SYM2677 and the ®rst eight bases in primer SYM2143 are linkers with no relevance for the application in thispaper.760 S. Lindquist et al. (Eur. J. Biochem. 269) Ó FEBS 2002ted with 50% formamide. After washing the ®lter, signalswere visualized using Molecular Imager (Bio-Rad).32P-Labelled k HindIII DNA was r un in parallel as a sizemarker.Cloning and DNA sequencingThe region o f r epeats in BSSL exon 11 was PCR ampli®e dusing 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 ddedto each reaction to a ®nal concentration of 2M(Sigma,St Louis, MO, U SA). A pair of primers, referred to asBSSL 12 and BSSL 14, was designed to cover the entiresequence of repeats (BSSL 12: 5¢-ACCAAC TTCCTGCGCTACTGGACCCTC-3¢;BSSL14:5¢-GGAGCCCCTGGGGTCCCACTCTTGT-3¢). The P CR started withadenaturationstep(96°C, 5 min) followed by 35 cycleswith 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 andthe fragments to be cloned were recovered using Gene-cleanII (BIO 101, Carlsbad, CA, USA). Cloning was p erformedusing t he pGEM Ò-T easy vector system II (Promega Co.,Madison, WI, USA). Before ligation into the pGEMÒ-Teasy vector, the PCR fragments had to be modi®ed using theA-tailing p rocedure for blunt-ended PCR fragments, asrecommended by Promega.The cloned fragments were sequenced on both strandsusing t he Big Dye terminator kit (PE Applied Biosystems,Foster City, CA) supplemented with betaine to a ®nalconcentration of 1M(Sigma). BSSL 12 or BSSL 14(described above) were used as primers, and t he DNAwas a mpli®ed for 30 cycles with denaturation a t 9 8 °C(30 s)and annealing/elongation at 60 °C (5 min). The reactionswere analyzed on an ABI PRISM 377A DNA sequencer(PE Applied Biosystems).RESULTSExpression of different BSSL variants in human milkTo con®rm the described heterogeneity in m olecular m ass o fmilk derived BSSL and select representatives for differentBSSL phenotypes we screened milk samples from ninedifferent mothers. The m ilk proteins were separated onSDS/PAGE, electroblotted and immunostained with BSSLspeci®c antibodies (Fig. 1). The most c ommonly occurringvariant of BSSL migrated with an appar ent molecular massof  120 kDa (donors D11, D8, D7). A variant with anapparently lower m olecular mass, i.e.  100 kDa, wasfound in some milk samples, either as the only on e (donorD2) or coexpressed with a variant of the most commonmolecular 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 alsocarried the 100 kDa variant in her milk. Donors D4 and D5carried only the 120-kDa BSSL variant in their milk samples(data not shown).Analysis of BSSL transcripts in milk cellsNorthern blot hybridization was performed on RNAisolated from m ilk from four different mothers, D1 andD6±D8 ( Fig. 2). A 2.8-kb transcript was detected in RNAfrom mother s D7 a nd D8 when a fragment c omplementaryto a sequence immediately upstream t he repeats i n exon 1 1was used as a probe (probe A; Fig. 3). A slightly shortertranscript,  2.7 kb, was d etected in RNA isolated frommother D6. The RNA isolated from mother D1 containedtwo hybridizing transcripts, 2.7 and 3.0 kb in size, respec-tively. To e xc lude the possibility that probe A had failed t odetect any possible truncated BSSL mRNA we usedanother probe, complementary to exon 2 to exon 4 in theBSSL cDNA (probe B; Fig. 3). However, identical resultsas with probe A w ere obtained using probe B in t heNorthern blot (data not shown).Genetic variation occurs in exon 11 of theBSSLgeneTo explore the possibility that genetic rearrangement(s)within the BSSL gene might explain the occurrence ofFig. 1. Naturally occurring variants of the BSSL protein in human milk.Milk proteins from seven dierent dono rs (D6, D11, D1, D8, D3, D2and D7) were separated on a 10% SDS/PAGE and immunostainedwith BSSL speci®c antibodies. The molecular mass standards areshownontheright.Fig. 2. Northern blot analysis of total RNA from milk cells isolatedfrom four dierent mothers (D1, D6, D7 and D8). The RNA washybridized to a BSSL speci®c probe, P robe A. HindIII-cut k was u sedas the m olecular mass m arker (M).Ó FEBS 2002 Molecular mass variants of BSSL in human milk (Eur. J. Biochem. 269) 761molecular mass variants of B SSL, we isolated DNA fromeight of the mothers (D1±D8) and p erformed Southern blothybridizations (Fig. 4). PstI digested DNA was hybridizedto a p robe complementary to a sequence in BSSL exon 11(probe A; Fig. 3). According to the published BSSLsequence [7±10] this probe was expected to hybridize to a731-bp PstI fragment carrying all 16 repeats. Accordingly, a0.7-kb PstI fragment was detected in all DNA samplesisolated from mothers c arrying the most common variant ofBSSL in their m ilk, i.e. D3±D8. However, this 0.7-kb PstIfragment was not found in DNA from m other D2, carryingonly the low molecular mass variant in her m ilk. I nstead, D 2and also the other mothers carrying low molecular massvariants in their milks (D1, D3 and D6) carried a shorterPstI fragment (0.6 kb). The mother with a high molecularmass BSSL variant in her milk (D1), carried a longerhybridizing PstI fragment (0.9 kb) not detected in any otherDNA s ample. Also a third PstI fragment (0.7 kb) wasdetected in DNA from mother D1. In contrast to the 0.9and 0.6 kb fragments t his 0.7-kb f ragment did not correlatewith any BSSL protein variant in milk from mother D1.When the DNA samples were digested with Eco RI andhybridized to probe C (Fig. 3) the hybridizing fragmentscorresponded t o the products obtained with PstIdigestionand probe A (Fig. 4b). DNA isolated from donorsexpressing the most common BSSL variant in milk (D3±D8) yielded a 2.2-kb EcoRI fragment when hybridized toprobe C. A shorter fragment ( 2.1 kb) was detected in DNAisolated from donors c arrying the 100-kDa variant of BSSLin milk (D1±D3 and D6). In DNA isolated from motherD1, thre e EcoRI fragments were foun d to hybridize toprobe C (2.1, 2.2, and 2.4 kb, respectively).Several other appropriate restriction enzymes and DNAprobes were used to cover the entire BSSL gene, looking foradditional genetic rearrangements. However, no geneticvariation 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 carryingthe repeats in exon 11 of the BSSL gene.PCR ampli®cation, cloning and DNA sequencingof different BSSL allelesTo further characterize some of the rearrangements in BSSLexon 11, we used PCR to amplify the region carrying therepeats in DNA isolated from two mothers (D1 and D2)(Fig. 5). According to t he published sequence [7±10] a678-bp fragment was expected to amplify if all the 1 6 r epeats(33 bp each) is present and if there is no deletions orinsertions. The results of the PCR con®rmed the Southernblot 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 bythe ampli®cation of a fragment  0.9kbinsize.Alsoinconcert w ith d ata f rom Southern blot, a third fragme ntcorresponding to the size of the wild-type a llele (678 bp) wasdetected in DNA from D1.The 0.6-kb PCR fragments, expected to carry theproposed deletions, were cloned from each of t he samples(D1 and D2) and the DNA sequenced. When thesequences were aligned to the previously publishedDNA s equence, it was con®rmed that t he deletions hadoccurred 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 wasshown to carry a 98-bp deletion that changes the readingframe of the gene and predicts a premature translationalstop after 632 amino acids (Fig. 7). The sequence f rommother D2 was essentially identical to D1 except that onebasepair 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 eletionchanges the reading frame and predicts a new C-terminaltail (RAAHG). Besides the d eletions, t he seq uences of D1and D 2 w ere i dentical to the published sequence e xceptfor one base substitution that does not affect the proteinsequence (Fig. 6).TheBSSLgene contains a hypervariable regionin exon 11To estimate the frequency of the BSSL polymorphism in alarger population, DNA was isolated from 2 95 healthyblood donors, digested with PstI and hybridized to probeA (Fig. 3) in Southern blot experiments. A high frequencyof variation was found. Only 131 out of the 295 (44%)DNA samples showed a restriction pattern correspondingto the published sequence, i.e. a PstI fragment  731 bp insize. In 23 out of 295 ( 8%) analyzed DNA samples, a PstIfragment considerably shorter than 731 bp was detected.As many as 41% (121/295) o f the analyzed DNA samplesshowed a heterozygous pattern with one PstI fragment 731 bp in size and another fragment considerablyshorter. An increased length of the actual PstI fragmentwas found in 21 out of 295 (7%) of the analyzed DNAsamples.DISCUSSIONThe BSSL locus is known t o exhibit a high degree o fpolymorphism [23], but whether this polymorphism affectsthe BSSL coding region has not previously been shown.Therefore, in the present paper we have investigated if theFig. 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 andnumbered 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 2002occurrence of different BSSL variants in human milk is du eto genetic variation with in the BSSL gene.We collected milk samples and isolated RNA and DNAfrom nine lactating mothers. Southern blot hybridizationexperiments con®rmed the occurrence of allelic variance inthe BSSL gene. The variations were exclusively foundwithin a Pst I fragment covering a region of direct repeats i nexon 11, and in each woman a correlation to the molecularmass of BSSL in milk was evident. Mothers known to havelow molecular mass variant(s) of the BSSL protein i n theirmilk were shown to carry a deletion,  0.1 kbinsize,withinthis PstI fragment. Mothers with two differen t BSSLvariants in t heir milk, e.g. the most common 120-kDavariant together with o ne of l ower mass, carried the deletionin one of the alleles, whereas t he mother with only the lowmolecular mass variant in milk (D2) carried deletions inboth alleles. In DNA isolated from mother D1, known t oexpress a high molecular m ass variant (160 kDa) togetherwith a low molecular mass variant in her m ilk, PstIfragments of 0 .6 and 0 .9 kb were detected. The sizes of thesefragments c orrespond to a 0.1-kb deletion i n one allele, anda 0 .2-kb i nsertion in another a llele, and are likely to encodethe low and high molecular mass variants detected in milkfrom D1, respectively. However, a third, unexpected PstIFig. 4. Southern blot hybridization. (A) DNAisolated from d ono rs D1±D8 w as digestedwith PstI a nd h ybrid ized to probe A (T ab le 1,Fig. 3). Hybridizing fragments shorter t han0.56 kb correspond t o fragments within thepseudogene CELL. HindIII-cut k was usedas molecular m ass marker (M). (B) DN Aisolated from th e same donors a s above wasdigested with EcoRIandhybridizedtoprobeC(Table1,Fig.3).HindIII-cut k was usedas molecular m ass marker (M).Ó FEBS 2002 Molecular mass variants of BSSL in human milk (Eur. J. Biochem. 269) 763fragment, was detected in DNA from mother D1. Thisfragment was  0.7 kb, corresponding to a fragment sizeprobably carrying the most commonly occurring 16 repeats.This fragment probably originates from a duplication of theBSSL gene, or at least of the 5¢ end, in one of the alleles.This duplicated copy was not expressed as on ly two variantsof the protein were found in milk from this donor.A DNA fr agment covering the deletions in exon 11 wasPCR ampli®ed, cloned and sequenced from two differentmothers (D1 and D2). The DNA sequences con®rmed t hedeletions and the deduced amino-acid sequences predictedBSSL variants of considerably lower molecular m ass, i.e. thevariants were predicted to be truncated after 632 and 610amino acids, respectively. Hence, these BSSL variants aretruncated within the region of proline r ich repeats and thenumber o f repeats is decreased from 16 t o 8.5 and 6.5,respectively, in the D1 and D2 variants. In both variants, anew C-terminal sequence consisting of ®ve a mino acids(RAAHG) is created due to the delet ions. In the wild-typeprotein (the most common v ariant), the C -terminal consistsFig. 6. The DNA sequence of the rep eatedregion c arrying a de letion in exon 11 frommother D1 and D2 w as aligned t o the publishedBSSL sequence (w t). The repeats arenumbered 1±16 according to the wt sequence.Alignments were performed using theprogramBESTFITfrom the U niversity ofWisconsinGCGsoftware packa ge. Dotsrepresent gaps that were inserted to improvealignment. The position of primers 12 and 14used f or ampli®cation and sequencing of thefragments a re marked. A n asterisk ( *) marksthe position of the single base subs titutiondetected in D1 an d D2.Fig. 5. PCR analysis of BSSL exon 11. DNA f rom mother D1 and D2was ampli®ed using a pair of primers covering the entire region ofrepeats i n t he BSSL ge ne. Three independent reactions were run fromeach 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 2002of the 16 repeats followed by a hydrophobic tail of 11 aminoacids. The low molecular mass variants expressed bymothers D1 and D2 d id not react with speci®c antibodiesdirected towards t his tail ( M. StroÈmqvist, AstraZenecaR&D,MoÈlndal, Sweden, personal communication) con-®rming a different sequence of t he tail. The f unction of thetail has previously been discussed in the literature. Deletionof the tail by in vitro mutagenesis of the human enzyme wasshown to signi®cantly decrease expression of the protein,presumably by affecting mRNA s tability [16]. F rom studieson the crystal structure of bovine BSSL it was concludedthat the terminal six hydrophobic amino acids physicallyblock a putative oxyanion hole at t he active site. Calcula-tions indicated that removal of this hexapeptid e exposes alarge hydrophobic area on the protein surface suggestingthat displacement of this re gion can play a role in thestability and function of BSSL [34].The s ize o f the BSSL transcript has p reviously beenestimated to be  2.5 or 2.9 kb [7,9]. We detected BSSLtranscripts of 2.8 kb in Northern blots p erformed on RNAfrom two mothers (D7 and D8) known to have the mostcommon BSSL genotype and phenotype i n milk. RNAisolated fr om mother D1, expressing t he high molecularmass varian t together with a low molecular m ass v ariant ofBSSL in milk, carried two transcripts that hybridized to theBSSL-speci®c probe. The sizes of these two transcripts wereestimated t o b e 2.7 and 3 .0 kb, respectively. Accordingly, weexpected to ®nd two transcripts in RNA from mother D6,known to have two BSSL variants (100 + 120 k Da) i nmilk, and to carry t he exon 11 deletion in one of the BSSLalleles. However, only one transcript was detected. Theband representing this transcript is however broad and webelieve that the resolution of the gel was insuf®cient t oseparate the two proposed transcripts.The frequency of variation in exon 11 of the BSSL gen ewas determined by Southern blot experiments with DNAisolated from 295 blood donors. When compared to thepublished s equence [7±10] 56% of the individuals examinedcarried genetic variations within the repeats. These datacon®rm that BSSL is located in a hypervariable region [23]but also shows t hat the polymorphism is due to deletions orinsertions within the BSSL coding sequence. Hence, weconclude that exon 11 in th e BSSL gene consists of ahypervariable r egion and that the current understandingthat exon 11 of the human gene encodes 16 proline-richrepeats is an oversimpli®cation and needs to be revisited.This high frequency of variation in the BSSL genecorresponds very well with a previous study on incidenceof molecular forms of BSSL in human milk [29]. This studyshowed that 50% of the milk samples contained BSSLvariants with a molecular m ass different to the m ostcommon variant.An onco-fetal variant of BSSL, d enoted feto-acinarpancreatic p rotein (FAPP), has been d etected in humanembryonic and fetal pancreas and in pancreatic tumoralcell lines [35,36]. FAPP and BSSL are structurally closelyrelated, but are distinguished by a monoclonal antibodydirected towards a fucosylated epitope, present on FAPPbut not on BSSL [37]. Compared to BSSL, FAPP h aslower enzymatic activity against ester substrates, and ispoorly secreted [36,37]. The cDNA sequence of FAPP isidentical to that of BSSL except for a 330-bp deletion inthe C-terminal repeated region [38,39]. T he fact that wenow show that  50% of a Swedish population c arry adeletion in the r epeated region of the BSSL gene makes i ttempting to speculate that FAPP is identical to a naturallyoccurring low molecular mass variant of BSSL. Thecharacteristic FAPP epitope should then result from tissuespeci®c glycosylation, rather than structural features of theprotein. If so, th e concept of F APP being an onco-fetalvariant of BSSL, exclusively expressed in proliferating cellssuch as embryonic and fetal panc reas as well a s pancreatictumoral cells, should b e r e-evaluated. The human hepa-toma cell line HepG2 also expresses a BSSL isoform oflower molecular m ass [ 40]. The cDNA sequence of t hisisoform contained only one ÔrepeatÕ.The obvious question is, of course, whether there arebiological phenotypes associated with speci®c BSSL vari-ants? As mentioned above, it has been proposed that there isno signi®cant difference in enzymatic activity, bile saltstimulation, pH stability a nd temperature stability b etweenBSSL of the most common molecular mass and variants oflower or higher m ass [27,28]. However, some l ow molecularmass variants with only half the speci®c activity comparedto the most common variant have been isolated and theconcentration of BSSL was co nsiderably lower in milk frommothers carrying only low molecular mass variant(s) [28].A possible explanation of th ese somewhat contradictoryresults could be the presen ce o r absence of t he mostC-terminal 11 amino acids, re ferred to as t he tail. Two l owmolecular mass variants characterized in this paper werebothshowntolacktheÔnormalÕ C-terminal tail, whereasStroÈmqvist et al. [ 28] showed that the tail is p resent in otherlow molecular mass variants. From the crystal structure ofbovine BSSL the tail was suggested to be involved in theactive site machinery [34].Finally, a positive correlation has b een demonstratedbetween BSSL activity in serum, assayed as cholesterolesterase activity, and serum cholesterol levels [5,6]. More-over, in vitro BSSL was shown to transform larger LDLparticles to smaller, more atherogenic LDL particles [41].Considering the data presented in the present paper, it isinteresting to note that an association between BSSLgenotype and serum lipid levels has been suggested [24,25].Taken together, we have shown that the molecular massvariants of BSSL found in milk results from a polymor-phism in the BSSL gene. This strongly suggests that BSSLvariants described in other tissues, such as the onco-fetalprotein FAPP, is due to the s ame frequently occurringFig. 7. Comparison of the deduced amin o-acid sequences of the repeatsin B SSL from the published sequence carrying 16 repeats (w t), and theshorter v ariants from mother D1 and D2.Ó FEBS 2002 Molecular mass variants of BSSL in human milk (Eur. J. Biochem. 269) 765genetic variation. 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Human bile salt-stimulated lipase has a high frequency of size variation due to a hypervariable region in exon 11 Susanne Lindquist1, Lars BlaÈ ckberg2and. 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 healthyblood
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