Expression of lipoprotein lipase gene in combined lipase deficiency

’ Mediantic Research

unt Sinai School of Medicine, New Yor&, NY.

National Institule of Diabetes.andDigesriwand Kidng Diseases, National hstirrtres of Health, Bethesda, MD, and 6 San&z Research Insrimte, East Hanover, NJ (U.S.A.) (Receivd 15 November 1988) (Revised manuscript received 11 April 1989)

Key words: Lipoprotein iipase; Lipase deficiency; Lipase gene; (Mouse)

Combined hpase deficisncy (c/d) in mice is a furii;tional deficiency of both lipoprotein lipase (LPL) and hepatic tri@yceride lipase (HTGL) [l]. This recessive mutation localizes within the T/t complex ‘of mouse chromosome 17 [2,3]. The bomozygotes develop severe owed to suckle they hyperchylomicronemia and when die within 3 days because of diffuse microinfarctions. Although there is 2-6-times more LPL-like :protein in tissues of affected mice Uutn in unaffected mice [4]; only a small amount of LPL in cld mice is released into plasma aftrerheparin infusion [5]. The apparent molecular weight of LPL on SDSPAGE of affected mice tissues k similar to that in unaffected mice [4]. These (data suggest that LPL-like protein is synthesized in CM mice, but that it may not be fully processed to express --Abbreviations: IkL, lipoprotein LipilSe(EC 3.1.1.34); HTGL, hepa.tic triglyceride lipase (EC 3.1.1.3); TE, Tris/EDTA buffer; RER, rough endophwnic reticuhun; cld, cornbmvxl lipasedeficient. Correspondence: K. Gka, Medhmtic Research Foundation, 108 Irving Street. N-W., Washington, DC 20010, U.S.A.

the enzyme activity. However, the expression of the LPL gene in cld and unaffected mice has not been studied at the level of mRNA. The mice used in this study were rais& and bred at the laboratory of Cellular and Developmental Biology of the National Institutes of Diabetes, and Digestive and Kidney Disease, Bethesda, MD. In back breeding studies, the cld gene has been linked to a tailless marker. Mice which had no tails and whose blood was hyperlipemic were classified as homozygous for cfd [l]. Mice with normal or short tai!s and no apparent hyperhpemia were classified as unaffected controls and included both homozygotis wild types and heterozygotes. l-day-old mice were killed by decapitation and tissues were excised and frozen immediately in liquid nitrogen. DNA and RNA were isolated by ~ornog~~ng in 6 guanidium isothioqanate/SO mM EDTA/SO mhd TrisNC1 (pH 7.5)/0.5% sodium sark(Jsyl followed by uhaacenuifugation on a 5.7 M CsCl cusbiotm [6]. The RI”:?E, was recovered from the pellet by dissolving in tissue resuspension buffer (5 mM EDTA/O.S% sarkosyW% /3-mercaptoethanol). The RNA solution was then ex-

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352 tracted with phenol/chloroform and chloroform, precipitated with ethanol, and stored at - 2 0 ° C until use. The CsC! solution containing DNA was diluted with an equal volume of sterile water, and the DNA was precipitated with ethanol. The DNA was recovered by spooling the stringly white DNA precipitate on a pipette, and rinsed with 70% ethanol. The D N A was resuspended in TE buffer, extracted with phenol/chloroform and chloroform, precipitated with ethanol, and stored at --20°C until use. The concentrations of DNA and RNA samples were evaluated by the absorbance at 260 nm with A26o/A280 m 2. RNA blot analysis [7] was performed on total RNA prepared from mouse tissues to detect LPL-specific mRNA. 20 pg of total RNA extract from tissues of affected and unaffected mice were prepared in 40 mM Mops (pH 7.0)/10 mM sodium acetate/1 mM EDTA/6% formaldehyde/50% deionized formamide, incubated at 65 ° C for 5 min, and chilled on ice. 1 ml of sequencing buffer was added and then electrophoresis was performed in a 1.2% agarose/6% formaldehyde gel. After electrophoresis, the gel was soaked in sterile water and KNA was transferred to a nitrocellulose filter (Schieicher & Schuell, NH). The RNA was fixed on the filter according to the manufacturer's protocol, and hybridized in 5~ dextran sulfate/25 mM potassium phosphate buffer (pH 7.4)/5 × SSC/5 x Denhardt's sohition/l% SDS/denatured salmon sperm DNA (100 pg/mi) at 65 ° C. The probe used was the 1575 bp EcoRl fragment of hLPLT-2 cloned from human placenta eDNA library [8] using bovine LPL cDNA as a probe [9], and labeled by random priming [10]. The DNA sequence of this fragment is identical to the basis from 64 to 1638 of human adipose tissue LPL cDNA reported by Wion et al. [11], which contains the entire open reading frame. The filter was then washed in 1 x SSC/0.1% SDS at 50°C, and exposed to Kodak XAR-5 film. To determine whether there is an LPL gene mutation in old mice, the genomic DNAs were evaluated by Southern blot analysis. 5 pg of genomic DNA prepared from tissues of cld and unaffected mice were digested with various restriction endonucleases at 5 units/pg DNA. The digested DNAs were electrophoresed in 0.7-1.2% agarose gel depending on the enzyme used, and transferred to a Hybond N nylon filter (Amersham, IL) in 10 × SSC according to the method of Southern [12]. The DNA was fixed by heating at 80°C for 2 h. The filter was pretreated and hybridized with the human LPL probe in 6 × SSC/5 × Denhardt's solution/0.5% SDS/denatured salmon sperm DNA (20 pg/ml) at 65 o C. The filter was then washed in 1 × SSC/0.1% SDS at 65°C, and exposed to X-ray film. As shown in Fig. 1, LPL-specific mRNAs with molecular sizes of 3.4 and 3.6 kb were detected irL defective and unaffected animal tissues, as reported by Kirchgessner et al. [13]. However, the concentration of

Fig. 1. Transfer blot analysis of total RNA from mouse tissues. 20 Itg of total RNA extracted from brown adipose tissue of old mice (1) and unaffected mice (2), and livers of old mice (3) and unaffected mice (4) were electrophoresed in a 1.2~ agarose/6~ formaldehyde gel and blotted onto a nitrocellulose fdter (Schleicher & Schuell, NH). The RNA was fixed on the filter by baking at 80 o C for 2 hr. in a vacuum oven and hybridized with a 32p-labeled EcoRI D N A fragment of hLPL7-2 in 5~ dextran sulfate/2.5 mM potassium phosphate buffer (pH 7.4)/5 × SSC/5 × Denhardt's s o l u t i o n / l ~ SDS/100 pg/ml of denatured salmon-sperm D N A ~t 65 o t.. The f'dter was then washed in I × S S C containing 0.1~ SDS at 5 0 ° C and exposed to Kodak XAR-5 film. The arrows indicate mouse 28 S and 14 S RNA.

LPL mRNA in affected animal tissues was higher than that in unaffected animal tissues. The same blot was rehybridized by b o ~ n e galactosyltransferas¢~ c D N A [14]. Although the m R N A was detected more in adipose tissues than in the livers, no difference of the size and amount was observed between c!d and unaffected animals (data not shown). This suggests that the increased LPL-like protein mass measured by immunoassay in old mice is due to an increase in transcription of LPL-specific mRNA. The identifiable defect in the mutant animal is the lack of enzyme activity [1,4] and the ina~ifity to be released by heparin [5]. Since the molecular weight is apparently unaffected [4], the defect must be subtle if it is within the reading frame of the LPL gene. O~te possibility is that there is a gene mutation which may alter an amino acid at or near the active site [11], the heparin binding site ['15] or the domain important for organelle transport. If this is the case, a nucleotide substitution may be detected by Southern blot analysis, following cleavage of genomic DNA with various restriction endonucleases, when the mutation exits within the sites recognized by the an-

353

Fig. 2. Genomic blots of D N A obtained from cld mice (lanes 2, 4, 6, 8) and from normal mice (lanes 3, 5, 7. 9). 5/~g genomic DNA were digested with BamHl (lane 2, 3), MspI (lanes 4, $), XhoI (lanes 6, 7) and Pstl (lanes 8, 9) at 5 units per pg DNA followed by electrophoresis in 0.7% ay~ose gel. D N A was transferred to a nylon fiher according to the method of Southern [12] and baked at 80 o C for 2 h to fix DNA. The fi|ter was thegn pretreated and hybridized to a labeled 1575 bp fragment of hLPL7-2 in 5 × SSC/5 × Denhardt's solution/0.5% SDS/20 p g / n d of denatured salmon-sperm D N A at 65°C. The filter was then washed and exposed to X-ray film at - 7 0 o C with intensifying screens. Lane 1 shows lambda HindIH molecular size standard labeled by [ 3sS]dATP.

zymes employed. Using this method we an~dyzed genomic DNAs. The following enzymes were used: Accl, Awfll, BamHl, BgllI, EcoRI, HaelH, Hi~dIII, Kpn|, MspI, Pstl, Pvull, SphI, Sstl, TaqI, XbaL and Xhol. None of these enzymes showed a restriction fragment lenff;h polymorphism (RFLP) between cld and unaffec';ed mice (Fig. 2). The cDNA probe used contains the entire open reading frame for human LPL, and the amino-acid sequence is highly conserved eetween animal species [13,15]. Therefore, the inability to detect a RFLP cannot be attributed to the use of human LPL cDNA. However, the possibility of a point mutation may not be exluded, because, only a small percentage of point mutations can be detected by this me~hod. The molecular size of the LPL protein in cid mice is not ~fferent from that in a,~fec:~ .,~2~e [4], and the present results indicate that the size of the mRNA is similar in both animals. The lack of demonstration of a

defect in the LPL gene or its expression in the RNA or protein in cld mice suggests that combined lipase deficiency may be caused by defective processing common to LPL and HTGL rather than to a structural mutation of either or both enzymes. Supporting this possibility is the fact that both LPL a~Jd HTGL are aff~ted, but these genes have been mapped on two separate chromosomes 8 and 9 [16!o One attractive hypothesis that would be c3nsistent with the present results is that defective protein glycosy|ation is involved. Mutatr.~ns within the T / t complex of mouse chromosome 17 are known to influence protein Slycosylation [17,18]. Mouse LPL iis a glycoprotein [13], and glycosylation has been reporl~ed to be essential for the expression of the enzyme activity in 0b 17 cells [19]. In addition, irnmunofluorescence analysis indicated that LPL-like protein in cld mice is accumulated in the RER [20], whereas normal LPL is secret~ [21]. Therefore, the actual lesion in cld

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mice may be restricted to the events within the RER after translation, or to the transport of LPL protein from the RER to the Golgi apparatus. The authors wish to thank L.A. Houghton and G.T. Tkalcevic for the technical assistance and S. Mody for the preparation of the manuscript. This work was supported in part by NIH grant HL32144. Ref.~rences 1 Paterniti, J.R., Jr., Brown, W.V., Ginsberg, H.N. and Attzt, K. (1983) Science 221, 167-169. 2 Bennett, D. (1975) Cell 6, 441-454. 3 Artzt, K., McCormick, P. and Bennett, D. (1982) Cell 28, 463-470. 40livecrona, T., Chemick, S.S., Bengtsson-Olivecrona,G., Patemiti, J.R., Jr., Brown, W.V. and Scow, R.O. (1985) J. Biol. Chem. 260, 2552-2557. 50fivecrona, T., Bengtsson-Ofivecrona,G., Chernick, S.S. and Scow, R.O. 41986) Biofhim. Biophys. Acta 876, 243-248. 6 Chirgwin, J.M., Przybyla, A.E., MacDonald, R.J. and Rutter, W.J. (1979) Biochemistry 18, 5294-5303. 7 Thomas, P.S. (1980) Prof. Natl. Acad. Sci. USA 77, 5201-5205.

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