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marker (1 23 bp, BRL) was applied. reveal any difference as compared to the published cDN A sequence [9,11,12]. The nucleotide sequence of exon 5, how-.
h r . J . Riochem. 208,267-272 (1992)

(g>FEES 1992

A missense mutation Prol57Arg in lipoprotein lipase (LPLNijmegen) resulting in loss of catalytic activity T x o BRUIN', John J. P. KASTELEIN', Denise E. VANDIERMEN', Yuanhong MA4. Howard E. HENDERSON4, Paul M. J. STUYT', Anton F. H. STALENHOEF', Auguesk STURK John D. BKUNZELL3 and Michael R. HAYDEN4

',

Centre for Hemostasis, Thrombosis, Atherosclerosis and Inflammation Rescarch, Academic Medical Centre, Univcrsity of Amsterdam, The Nethcrlands Division ol' General Internal Medicine, University Hospital Nijmegen, Thc Netherlands Division of Metabolism, Endocrinology and Nutrition, Department of Medicinc, University of Washington, Scattle, USA Department of Medical Genetics, University of British Columbia, Vancouver, Canada

(Keceived (April 9/June 9, 1992) - EJB 920502

Here we report on the molecular defect that leads to a deficiency of lipoprotein lipase (LPL) activity in a proband of Dutch descent. Southern-blot analysis of the LPL gene from the patient did not reveal any major DNA rearrangements. Sequencing of polymerase-chain-reaction-amplified DNA revealed that the proband is a homozygote for C725C, resulting in a substitution of Pro157 for Arg. This substitution alters a restriction site for PvuII, whch allowed rapid identification of the mutant allele in family members. Site-directed mutagenesis and transient expression of the mutant LPL in COS cells produced an enzymatically inactive protein, establishing the functional significance of this mutation. This naturally occurring mutation which alters the Pro1 57 adjacent to Asp1 56 of the proposed catalytic triad, indicates that this region of the protein is indeed crucial for LPL catalytic activity.

Lipoprotein lipase (LPL) plays a pivotal role in the metabolism of triacylglycerols present in the core of chylomicrons and very-low-density lipoproteins (VLDL) [I]. LPL is a glycoprotein, active in its dimeric form [2]. Each protein chain consists of 448 amino acids and has a carbohydrate content of 3-10% by mass. Apart from interaction with heparan sulphate, LPL has at least five additional functional domains, including a catalytic site, a site for interaction with apolipoprotein C-11, a site for dimerization, a site for binding to the lipid/water interphase and a putative site for binding fatty acids [3 - 51. cDNA clones Tor the rat, bovine, guinea pig and human LPL gene have recently been isolated [6 - 91. LPL is a member of a gene family that includes hepatic lipase and pancreatic lipase [lo]. The gene for human LPL maps to chromosome 8p22 and comprises 10 exons, spanning approximately 35 kbp 111-131. LPL deficiency is inherited as an autosomal recessive trait and results in severe hypertriglyceridemia [l]. This disorder is characterized clinically by recurrent upper abdominal pain, pancrcatitis, hepatosplenomegaly, eruptive xanthomas and lipemia retinalis, often manifested in early childhood. The clinical manifestations subside with the restriction of dietary .

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fats and the resulting absence of postprandial chylomicronemia. More than 25 mutations in the LPL gene have, to date, been reported lo cause chylomicronemia, indicating that LPL deficiency is a genetically heterogeneous disorder. These mutations include a partial gene duplication, a deletion, an insertion and many missense mutations [14-251. The three-dimensional structure of human pancreatic lipase has facilitated the assessment of the catalytic triad in other members of the gene family [26]. Studies using sitedirected mutagenesis revealed that the proposed catalytic triad comprising Ser132, Asp156 and His241 in LPL is indeed essential for catalytic activity 127, 281. Moreover, we recently described two missense mutations in the codon for Asp156 [29], which supported its essential role in catalytic activity. In the present study, we have investigated the molecular defect in the LPL gene of a Dutch patient with LPL deficiency 1301. A missense mutation was found in exon 5 of the LPL gene, resulting in a substitution of Pro for Arg at position 157. This mutation is adjacent to one of the amino acids (Asp156) of the proposed catalytic triad and results in the loss of a restriction site for PvuII in exon 5 and allows for rapid screening for the mutant allele

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Correspondence to T. Bruin, Centre for Hemostasis, Thrombosis, Atherosclerosis and Inflammation Research, Academic Medical Centre, Room GI-I 14, Meibergdreef 9, NL-1305 AZ, Amsterdam, The Netherlands Ahhruviutions. PCR. polymerase chain reaction; RFLP. restriction-fragment-length polymorphism; HDL, high-density lipoproteins; LDL, low-density lipoproteins; VLDL, very-low-density lipoproteins. Enzyme. Lipoprotein lipase (EC 3.1.1.34)

MATERIALS AND METHODS Case history

The proband is a 32-year-old female with type-I hyperlipoproteinemia; her history has been described more extensively in an earlier report [MI. At the age of 6 weeks she underwent laparotomy because of a chyloperitoneum. At that

268 time, milky serum, hepatosplenomegaly and a chylothorax were noticed. A fat-restricted diet was prescribed and subsequently she grew up uneventfully. Plasma cholesterol levels varied over 5 - 8 mmol/l and triacylglyceride levels over 1030 mmol/l. Between the ages of 22 and 25 she was admitted to hospital because of pancreatitis on eight occasions. During five years of follow-up, triacylglyceride levels varied over 15 25 mmol/l and no further attacks of pancreatitis occurred. At the age of 25, a diagnosis of LPL deficiency was made. On isoelectric focussing of VLDL protein, a normal amount of apolipoprotein C-I1 was detected.

Analytical procedures In family members, total plasma cholesterol and triacylglycerides were measured by enzymatic methods (Boehringer-Mannheim) in a Cobas-Bio centrifugal analyzer (Hoffmann-La Roche). High-density lipoprotein (HDL) cholesterol was determined by measuring cholesterol in the supernatant liquid after precipitation of the apolipoprotein-B-containing lipoproteins with heparin and MnClz [31]. HDL and lowdensity lipoprotein (LDL) cholesterol levels were measured in the supernatant plasma after aggregation of VLDL with SDS 1321. VLDL and LDL cholesterol levels were then calculated from these values. Apolipoproteins AI and B were measured by fixed-time nephelometric assays in a BNlOO nephelometer (Behring Diagnostics).

Measurement of LPL activity and amount Plasma LPL activity in the proband and all family members was measured prior to and 10-min after the administration of an intravenous heparin bolus (50 Ujkg body mass). Patients were fasted for at least 12 h. Samples were cooled on ice and the plasma was separated from the cells after centrifugation (15 min, 2000 x g at 4"C), and stored at 70 "C. Total postheparin plasma lipolytic activity was measured as previously reported using a tri-[l-'4C]oleate as a substrate in a phosphatidylcholine emulsion 1331. LPL activity was calculated as the activity in whole plasma, inhibited by a specific monoclonal antibody added to plasma, and is expressed as nanomoles fatty acids released . f i n - ' . m l 'plasma-'. Values in 34 normal controls amount to 220 59 nmol . ml- . min- '. LPL, present in preheparin

'

plasma and released in postheparin plasma was measured by ELISA. Values in the normal controls were 196 & 59 ng/ml for the amount in post-heparin plasma [34].

DNA analysis Southern analysis

Genomic DNA was extracted from fresh EDTA-anticoagulated blood as described previously [ 3 5 ] . Aliquots of DNA (5 pg) from the initial proband and her family members were digested with the restriction endonuclease, PsA and other enzymes, that do not recognize restriction-fragment-length polymorphisms (RFLP) in the LPL gene, in order to screen for large gene rearrangements. RFLP were determined by digestion of genomic DNA with BamHI, HindIII, PvuII and BstI. These digested DNA fragments were clcctrophoresed through a 0.8% agarose gel and transferred to nylon membranes by the Southern-blotting technique [I 41. The human LPL cDNA clone, HLPL 26, was kindly provided by Dr. S. Deeb, University of Washington, Seattle. Polymerase-chain reaction and sequence anal-vsis

Genomic DNA from the proband and her family members was used for analysis by the polymerase-chain reaction (PCR). The reactions were performed in a Perkin-Elmer/Cetus corporation DNA thermocycler. The reactions for exon 5 were performed in the recommended buffer of Saiki et al. and contained 500 ng genomic DNA, 200 pM dNTP, 50 pmol each primer and 2.5 U Taq DNA polymerase (Amplitaq, Cetus corp.) in a final volume of 100 pl [36]. The final Mg2+ concentration was 1.1 mM. The reaction mixtures were denatured at 94°C for 1 min, annealed at 5 0 T for 1 min and extended at 72°C for 1 min for a total of 30 cycles. The oligonucleotide primers were synthesized in a DNA synthesizer (Gene Assembler Plus, Pharmacia). PCR-amplified exon 5 (100 pg) was digested with 10 U PvuII for 2 h at 37°C. These exon-5 fragments were electrophoresed through a 1.2% agarose gel and stained with ethidium bromide. The PCR-amplified double-stranded template was run on a 1.2% agarose gel and the amplified band was excised from the gel and purified using Gene Clean (Bio/lOl). Direct sequencing of the sense and the antisense strand of the tem-

Table 1. LPL-catalytic activity and amount, plasma triacylglyceride and lipoprotein concentrations in members of the LPL-deficient family. Column 2 gives the presence of the mutant allelc determined by PvulT digestion of exon 5 : 1, heterozygous; 2. homozygous: 3, normal. t = 0. values before heparin infusion; t = 10, values 10 min after heparin infusion. Control values for LPL activity and for LPL amount 10 mm after heparin infusion are 220 f59 nmol ml-I .min-' and 196 & 59 ng/ml, respectively. n.d., not determined.

Mutant

LPL

Concentration of

allele activity

1.1 1.2 11.1 11.2

11.3

1 1 1

2 3

148 128 305 3 n.d.

mass for t

0

10min

95 60 12 61 n.d.

249 183 278 82 n.d.

triacylglyceride

cholesterol

LDL

HDL

VLDL

apolipoprotein B

apolipoprotein A1

2.06 I .80 1.02 20.1 0.96

8.22 1.07 5.98 7.41 6.36

5.40 4.59 3.53 1.12 3.79

1.17 1.04 1.12 0.33 1.35

1.65 1.44 1.36 5.96 1.22

1.86 1.69 1.40 0.73 1.56

1.56 1.42 1S O 1.17 1.78

269 2

1

I

II

h

3;

111 2

+ -

Fig. 1 . Family tree showing haplotypes (BumHI, PvuII, Hind111 and BstI) and the presence (shaded circle or square) and absence of the mutant allele.

plate was performed using Sequenase-2 (United States Biochemicals) as described previously [37]. Site-directedmutugenesis and transient expression in COS cells A 1581-bp Drd-EcoRI fragment from a LPL cDNA clone, which contained the entire coding sequence, was cloned into a dual-function vector (pCDM8) for both mutagenesis and expression as previously described [29]. The phosphorylated mutant oligonucleotide (S-TCGATCGAGCTGGACCT-3’) was annealed to the single-stranded pCDM8 LPL DNA and second-strand synthesis was performed as previously described [29]. Mutant clones were identified by oligonucleotide hybridisation and verified by DNA sequencing. Transient expression of the mutant and wild-type control constructs in COS-1 cells was performed as described [21]. LPL activity and amount were determined in both media and cell homogenates. RESULTS LPL activity and mass LPL activity was determined in postheparin plasma for the initial proband and the other family members (Table 1).

Control

LPL activity in the proband was almost absent at 3 nmol . ml-’ . min- and mildly reduced in the parents, but normal in one sibling. The LPL deficiency in the proband therefore constitutes a catalytically defective protein. LPL (61 ng/ml) in the proband was present in pre-heparin plasma and increased only slightly after heparin infusion (82 ngiml), which may be due to an increased turnover of the mutant LPL or a decreased release of mutant LPL from parenchyma cells. Hepatic lipase activity (76 nmol . inl- . min-l) and apolipoprotein CIT levels were normal. Lipid and lipoprotein analysis An extensive lipoprotein profile and apolipoprotein levels were determined in the proband and all family members. The proband (Table 1) showed severe fasting hypertriglyceridemia, reduced LDL and HDL cholesterol levels and a reduced level of apolipoprotein B-100, compatible with the diagnosis of chylomicronemia syndrome (Table 1). Triacylglyceride levels were normal in all other family members. Southern-blot analysis Digestion with PvuII resulted in the detection of a largerthan-expected fragment in the proband (data not shown). Abnormal fragments were not seen with any other enzymes, which suggested that this was due to an altered restriction site and not to a major gene rearrangement. Haplotypes were constructed using the restriction enzymes BumHI, PvuII, Hind111 and BstI, with known polymorphic sites in the gene (Fig. 1). A search in the cDNA sequence of the LPL gene for PvulI sites, CAGCTG, revealed this recognition site in exon 2 and exon 5. To distinguish between a restriction-site loss in exons 2 or 5, a further analysis by PCR and direct sequencing of these exons of the proband LPL gene was performed.

PCR and sequence analysis Oligonucleotides homologous to intron sequences flanking exons 2 and 5 were used as primers to amplify the coding sequences of the gene from genomic DNA of the proband. Direct sequencing of exon 2 after amplification did not

Patien t

G A

h

G

\I f C G A T

G ~ J

C G A T

Fig. 2. DNA sequence of the normal and the mutant exon 5 showing a G to C change at position 725 results in the substitution ProlfArg. DNA sequence analysis was performed by electrophoresis on a 6% acrylamide/8 M urea sequencing gel. The figure gives the sequence of the antisense strand (3’+5’).

270

120 14"

1

(225 2 )

(447.0)

369 246-

123Ccll Homogenat?

Fig. 3. Family tree, showing loss of a PsuII recognition site in exon 5 of the LPL gene, due to the missense mutation. Exon 5 was amplified by PCR and digested with Pvull. This results normally in fragment s i x s of 247 bp and 38 bp, respectively. The missense mutation at residuc 725 abolishes a PvuII rccognition site, resulting in a single fragment of 285 bp. On thc left and right site of the gel a DNA size marker (1 23 bp, BRL) was applied.

reveal any difference as compared to the published cDN A sequence [9,11,12]. The nucleotide sequence of exon 5, however, showed a single-basepair substitution at nucleotide 725, when compared to the normal coding sequence of exon 5, where a G was substituted for a C (Fig. 2). The reading frame, therefore, was no1 altered, but this single basepair substitution predicts a missense mutation with a substitution of Arg at position 157 for Pro. Exon 5 was PCR amplified and subsequently digested with Pvull. PCR of exon 5 of the proband and relatives was subjected to agarose electrophoresis. Control DNA showed the predicted fragment sizes of 247 bp and 38 bp. However, the proband showed only a 285-bp band, indicating the loss of both PvuII recognition sites in exon 5. Both parents and a sibling proved to be heterozygous for this basepair change and another sibling proved to be normal (Fig. 3). The remaining exons of the gene were amplified by PCR and sequenced as described previously; no other DNA alteration was detected. An additional 50 unrelatcd homozygous LPL-deficient patients of Canadian, Columbian, Dutch, German, Maroccan, South African and Turkish descent were screened for the amino-acid-I 57 mutation, but none was found. Furthermore, the screening of an additional 100 alleles in the normal Dutch population did not reveal the mutation. The data from the haplotype analysis using PvuTI, HindIII, BurnHI and BstI are in accordance with the results from the PvuII digestion of PCR-amplified exon 5 in all family members (Fig. 1, 3 ) .

MrYi1um

b X of normal

120 (11.4)

Cell lloinoqenatr

(124.7)

hf r d 1 11 m

Fig. 4. LPL activity and amount in culture medium and cell homogenate of COS-I cells transfected with normal LPL cDNA and cDNA with the Prol57Arg substitution. Results are expressed as percentages of the levels determined in thc medium and cell homogenate of COS-I cells transfected with the normal LPL cDNA. (a) Amount of normal LPL (black column) and mutant LPL (striped column). (b) Activity of normal LPL (black column) and mutant LPL (striped column).

present in the medium as well as in the cell homogenate of COS cells transfected with the mutant Prol 57Arg cDNA (Fig. 4A). However, the mutant LPL was present at a slightly higher concentration in the cell homogenate and at a slightly lower concentration in the medium than in the control experiment. LPL enzymatic activity in the medium, as well as in the cell homogenate of the COS cells transfected with the mutant Prol 57Arg cDNA, was completely absent compared to control cDNA (Fig. 4B). This indicates that the mutant Pro157Arg cDNA indeed produces a catalytically inactive protein.

In-vitro expression of wild-type and mutant LPL

DlSCUSSION

In order to prove that the Prol57Arg mutation causes LPL deficiency in the proband, we introduced the mutation into cloned LPL cDNA by site-directed mutagenesis of the LPL-expression phagemid (pCDM8-LPL) using the mutagenic primer 5'-TCGATCGAGCTGGACCT-3'. We transfected COS-1 cells with both wild-typc and the mutant LPL cDNA Prol 57Arg expression phagemid. LPL protein was

This study demonstrates that a C to G (C725G) substitution at nucleotide 725 in exon 5 of the LPL gene results in a substitution of Pro for Arg at residue 157 (Prol57Arg) and produces a catalytically defective protein. Our data strongly suggest that this nucleotide substitution in exon 5 is the cause of LPL deficiency in this kindred. The original proband was hoinozygous for this nucleotide substitution and the C725G

27 1 1.

b132 LPL

2.

3.

Val-His-Leu-Gly-Tyr-Ser132 -Leu-Gly-Ala-His

HL

Val-His-Leu-Gly-Tyr-Ser

-LeuGly-Ala-His

PL

Val-Glu-LeuGly-His-Ser

-Leu-Gly-Aln-His

b156

LPL

Thr-Gly-Leu-Asp156 -PRO-Ala

HL

Thr-Gly-Leu-Asp

-Ala-Ala

PL

Thr-Gly-Leu-Asp

-Pro-Val

a241

LPL

Lys-Cys-Ser-Hls24 1 -Glu-Arg-Sei

HL

Lys-Cys-Ser-His

-Glu-Arg-Ser

PL

Ala-Cys-Asn-His

-Leu-Arg-Ser

Fig. 5. Similarities for the three amino acids of the catalytic triad in different members of the lipase family concluding LPL, HI, and PL.

substitution was not present in 100 alleles from normolipidemic controls. In addition, the mutation cosegregated with the expression of LPL deficiency in this family. Furthermore, we performed site-directed mutagenesis experiments using a COS-cell expression system to reproduce the Prol 57Arg substitution in vitro. Indeed, the mutant Prol 57Arg cDNA lost its enzymatic activity, while the protein was present at normal levels. The question remains as to why a single Pro157Arg substitution reduces the catalytic activity of LPL so dramatically. Proline frequently causes bending of protein chains and its cyclic structurc markedly influences protein tertiary structure. The substitution of a n aliphatic amino acid (Pro) for a positively charged amino acid (Arg) is likely to alter the spatial conformation of the LPL. The three-dimensional structure of human pancreatic lipase has been deternlined recently [26]. The authors proposed that the active lipolytic site in this enzyme is formed by a trypsin-like catalytic triad, Serl52His263-Asp176 which corresponds to Serl32-His241-Aspl56 in human LPL (Fig. 5). The N-terminal domain of human pancreatic lipase containing the active site [26] shows considerable sequence similarity to lipoprotein lipase, whose structure is likely to be very similar in the active-site region. Further evidence for this hypothesis is that LPL from human, rat, cow and guinea pig shows a high degree of interspecies similarity, but the amino acid sequences surrounding the three amino acids of the catalytic triad exhibit almost complete conservation. Almost complete similarity around these three amino acids is also observed between different members of the lipase family (Fig. 5 ) [lo]. This suggests that these sequences are of vital importance for normal LPL function. The proximity of Pro1 57 to a n essential residue suggests that any mutation disturbing the local structure could reduce the activity of the enzyme. This was also confirmed by the finding that both Asp1 56Asn and Asp156Gly mutants cause catalytically inactive LPL [29]. In the structure of human pancreatic lipase, this Pro (Pro1 77) is completely huried and is surrounded by the side chains of Ala155, His156, Leu191, Va1201, His203, Met217 and Val221 (F. Winkler, unpublished results). The corresponding residues in LPL are Alal35, His136, Leul71, Va1181, His183, Ile196 and Va1200, suggesting an almost identical packing in this region. A large and positively charged Arg side chain at the same position cannot be accommodated without considerable disturbance of the

local and perhaps even the tertiary structure of this domain, resulting in complete loss of catalytic activity. In summary, we present evidence that the G725C substitution in the LPL gene of a chylomicronemic patient substitutes Pro157 for Arg. This amino acid substitution will significantly alter the three-dimensional structure of the protein, leading to a catalytically inactive LPL enzyme. The authors wish to thank C. Reitsma-Vlaar for the preparation of this manuscript. We want to thank Dr. F. Winkler for his valuable suggestions. Dr. A. Stalenhoef is clinical investigator of the Dutch Heart Foundation. Dr. M. R. Hayden is an established investigator of the British Columbia Children’s Hospital and a gator of the Canadian Genetic Diseases Network. Dr. Y. Ma is a postdoctoral fellow supported by the Medical Rcscarch Council of Canada.

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