ELSEVIER Biochimica et Biophysica Acta 1302 (1996) 236-240

BB Biochi~ic~a et Biophysica AEta

Pancreatic lipase-related protein 2 but not classical pancreatic lipase hydrolyzes galactolipids

O

Lena Andersson a, Fr rd r r i c Carfi~re b, Mark E. Lowe c, Ake Ni lsson a,* , Rober t Verger b

a Gastroenterology Division, Department of Internal Medicine, Universi~' of Lund, S-221 85 Lund, Sweden b Laboratoire de Lipolyse Enzymatique UPR 9025, Centre National de la Recherche Scientifique, 13402 Marseille cedex 20, France

c Division of Gastroenterology and Nutrition, Department o f Pediatrics, Molecular Biology and Pharmacology, Washington Unit~ersi~' School of Medicine, St. Louis, MO 63110, USA

Received 19 January 1996; accepted 1 April 1996

Abstract

The pancreatic lipase family contains three subfamilies, the 'classical' lipases and the pancreatic lipase-related proteins 1 (PLRP1) and 2 (PLRP2). Galactolipids are present in membranes of leaves and vegetables and consist of digalactosyldiacylglycerol (DGalDG) monogalactosyidiacylglycerol (MGalDG) and sulfoquinovosyldiacylglycerol (SQDG). These lipids were incubated with PLRP2 from guinea-pig (GPLRP2) and rat (RPLRP2). In the presence of bile salts DGalDG was efficiently hydrolyzed by GPLRP2 and, although less efficiently, by RPLRP2 to digalactosylmonoacylglycerol (DGalMG), free fatty acids and water-soluble galactose-containing compounds. Also, MGalDG and SQDG were hydrolyzed by GPLRP2 and RPLRP2. These data suggest a possible role of PLRP2 in the digestion of dietary galactolipids.

Keywords: Digalactosyldiacylglycerol; Digestion; Galactolipid; Monogalactosyldiacylglycerol; Pancreatic lipase-related protein; Sulfoquinovosyldiacyl- glycerol

1. Introduct ion

The specificity of the lipolytic enzymes of the pancreas is well characterized for most kinds of lipids [1]. The role of different lipolytic enzymes in the digestion of galacto- lipids has, however, not been characterized. Galactolipids are mostly found in the thylakoid membranes of the chloroplasts but are also present in fruits and roots [2]. About 77% of the lipids in thylakoid membranes are neutral galactolipids dominated by digalactosyldiacylglyc- erol ( D G a l D G ) and monoga lac to sy ld i acy lg lyce ro l (MGalDG), a smaller part being sulfoquinovosyldiacyl- glycerol (SQDG). A man might consume an average of 150-250 mg galactolipids a day and the guinea-pig 500 - 900 mg a day. Our previous investigation showed that galactolipids were well hydrolyzed by human pancreatic juice or duodenal content generating digalactosyl monoa- cylglycerol (DGalMG), non-polar lipids, mainly fatty acids and water-soluble galactose-containing compounds as products [3]. Pure human pancreatic carboxyl ester lipase

* Corresponding author. Fax: +46 46 137277.

0005-2760/96/$15.00 © Elsevier Science B.V. All rights reserved PII S0005-2760(96)00068-9

(CEL, EC 3.1.1.13) had galactolipase activity and hydro- lyzed MGalDG and SQDG better than DGalDG. Purified human pancreatic lipase (HPL, EC 3.1.1.3) together with colipase hydrolyzed MGalDG at a low rate but had almost no activity against DGalDG or SQDG [3].

When pancreatic juice was separated on a Sephadex G100 column, activity was found towards all three galacto- lipids in two peaks. The smaller one comigrated with CEL and the peak with highest lipolytic activity comigrated with HPL [3]. The results thus suggested that an enzyme with galactolipase activity is present in pancreatic juice with a molecular mass similar to HPL, but which is not identical to this enzyme.

A possible explanation for our findings is suggested by the recent description of two new members of the pancre- atic lipase family, pancreatic lipase-related proteins, HPLRP1 and HPLRP2 [4]. Based on amino acid sequence comparisons, Giller et al. [4] classified the pancreatic lipases into three subfamilies. Subsequently, members of each subfamily have been cloned from different species [5-7]. Both human and rat PLRP1 have been expressed and characterized, but no significant activity has been

L. Andersson et al. / Biochimica et Biophysica Acta 1302 (1996) 236-240 237

detected with either triglyceride or phospholipid substrates [4,6]

In contrast, the PLRP2 from guinea-pig (GPLRP2, Ref. [7]), coypu (CoPLRP2, Ref. [5]), and rat (RPLRP2, Ref. [8]) possess high lipolytic activity towards both phospho- lipids and triglycerides, a property that separates them from classical pancreatic lipase which prefers triglycerides. Using physiological concentrations of bile salt in the assay, GPLRP2 and CoPLRP2 lipase activity is inhibited like the classical pancreatic lipase, but such activity is not restored by the addition of colipase as is the case with the classical pancreatic lipase. RPLRP2 differs from GPLRP2 and CoPLRP2 since it is not inhibited by physiological concen- trations of bile salts, but, like the other PLRP2 lipases, RPLRP2 is not dependent on colipase for activity [8]. The physiological substrate for the PLRP2 lipases is not known. In fact, it has been proposed that GPLRP2 and CoPLRP2 might only hydrolyze phospholipids in vivo because guinea-pig and coypu pancreas produce a classical pancre- atic lipase [9,10] and no detectable phospholipase A 2 (EC

3.1.1.4) activity [5,11]. In rats and in humans, the phospho- lipase A 2 is present and PLRP2 might play another physiological role.

The aim of the present study was to investigate whether the novel pancreatic lipase-related proteins were active towards galactolipids.

detector SPD-6A at 205 nm) equipped with a Nucleosil 50-5, 250 × 46 mm colon using a mobile phase of propan- 2-ol/hexane 4:3. [3H]UDP-Galactose (NET-758, 30-50 Ci/mmol) was obtained from Du Medical Scandinavia, Sweden to be used for the synthesis of [3H]galactose- labelled DGalDG by a method based on studies of galac- tolipid synthesizing enzymes in pea seedlings by Siebertz et al. [14] and Andersson et al. [3].

2.3. Incubation conditions

All incubations were performed in 10 mM Tris-maleate buffer at pH 7.0 containing 2 mM CaC12 and 0.12 M NaC1 with 6.4 mM bile salt mixture containing 42% (w/w) NaTC, 26% NaTDC, 22% NaGC and 10% GCDC or amounts indicated in legends to figures. Substrate concen- trations of 2 m g /m l was dispersed in buffer with bile salts by sonicating for 2 × 30 s output 20% with a Branson Sonifier 250 (KEBO lab, Sweden) and preheated to 37°C. The incubations were started by adding different enzymes at final concentrations of 0.5, 2 or 5 I~g/ml. Reactions were terminated at different time intervals during a 2-h incubation either by heating on a boiling waterbath for about 5 min (experiments where FFA were determined enzymatically) or by extracting with chloroform/methanol 1:1 (experiments with radioactive substrates) [15].

2. Materials and methods 2.4. Determination of galactolipid hydrolysis

2.1. Sources of pancreatic lipase-related proteins and bile salts

Rat recombinant PLRP1 without any enzyme activity for triglycerides or phospholipids, and rat-PLRP2 with phospholipase and lipase activity was purified as described by Payne et al. [6]. Recombinant GPLRP2 with a specific activity of 2000 U/mg, using tributyrin as substrate, was expressed in Aspergillus orizae and purified as previously described [7]. Sodium-taurocholate (NaTC), sodium tan- rodeoxycholate (NaTDC), sodium glycocholate (NaGC) and sodium glycohenodeoxycholate (NaGCDC) were syn- thesized by the method of Norman [12] (by Dr. Lennart Krabisch, Dept. of Physiological Chemistry, University of Lund).

2.2. Sources of galactolipids

Chromatographically pure DGalDG, MGalDG and SQDG, prepared from plant leaves, were obtained from Lipid Products, S. Nutfield, Surrey, UK. [3H]Fatty acid labelling of galactolipids was performed by Amersham Tritium Labelling Service, UK and purified into individual galactolipids by thin layer chromatography as described by Bratt et al. [13]. [3H]Fatty acid-labelled MGalDG was further purified by HPLC (Shimadzu LC-6A with UV

2.4.1. Rate of hydrolysis We determined the rate of hydrolysis by measuring the

amount of free fatty acids released during incubations with NEFA-C kit (Wako Chemicals, Germany), an in vitro enzymatic method for the quantitative determination of non-esterified fatty acids.

2.4.2. Hydrolysis of radioactive galactolipids When [3H]galactose-labelled DGalDG or [3H]fatty

acid-labelled DGalDG was used as substrate, the reaction was interrupted by extracting aliquots of the incubation mixture with chloroform/methanol 1:1 [15] containing 0.005% butylated hydroxytoluene at the time intervals indicated in Fig. 2a-c. Aliquots of the lipid extracts were separated on TLC plates developed in chloroform/ methanol/acetic acid/water 60:40:3.0:0.3. Radioactivity of DGalDG, DGalMG, non-polar lipids and galactose-con- taining compound was determined as described earlier [3].

2.4.3. Characterization of the polar hydrolysis product of DGalDG, MGalDG and SQDG

After incubations of galactolipids with GPLRP2, lipids were extracted and separated by TLC. The spots were eluted from the TLC-plates. Half of each eluate was subjected to acid hydrolysis before free galactose was determined. The other halves of the eluates were subjected

238 L. Andersson et al. / Biochimica et Biophysica Acta 1302 (1996) 236-240

to alkaline hydrolysis before determining unesterified fatty acids by the NEFA-C kit [3].

3. R e s u l t s

To determine the hydrolysis of galactolipids by GPLRP2, RPLRP2 or RPLRPI we incubated unlabelled DGalDG, MGalDG or SQDG with GPLRP2 or RPLRP2 in the presence of bile salt concentrations indicated in the legends a -d of Fig. 1. By measuring the release of FFA during incubations we found that GPLRP2 and RPLRP2 hydrolyzed all three galactolipids. The hydrolysis rate was higher with GPLRP2 than with RPLRP2. At bile salt concentrations 4.8 mM and lower, the hydrolysis of DGalDG by GPLRP2 was slow, whereas bile salt concen- trations of 6.4 mM and higher increased the hydrolysis rates (Fig. la). The optimal activity for the hydrolysis of DGalDG with RPLRP2 was observed with 2 - 4 mM bile salts; higher concentrations inhibited the hydrolysis (Fig. lb). In incubations of different galactolipids with RPLRP1 with or without bile salts (0, 2, 6.4 mM), we were not able to demonstrate any hydrolysis.

To characterize the products from hydrolysis of galacto- lipids, we used pure unlabelled DGalDG together with [3H]galactose-labelled or [3H]fatty acid-labelled DGalDG. During incubations of [3H]galactose-labelled DGalDG with GPLRP2, there was a rapid decrease of radioactivity in DGalDG and an increase of 3H in DGalMG and water- soluble compounds (Fig. 2a).

After 20 min the amount of DGalMG formed was slightly higher than the amount of water-soluble products, but within 120 min most DGalMG was further hydrolyzed to water-soluble compounds, indicating that the enzyme hydrolyzes fatty acid esters in both positions (Fig. 2a).

In the incubations of [3H]fatty acid-labelled DGalDG with GPLRP2, the analysis of the hydrolysis products showed a decrease with time in the radioactivity of DGalDG spot and the appearance of a new, more polar product. The radioactivity in non-polar lipids also in- creased with time. After 120 min the enzyme had hydro- lyzed about 80% of the radioactive DGalDG, leaving 36% as DGalMG and 42% as non-polar lipids (Fig. 2b). In an alkaline TLC system (mobile phase chloroform/metha-

Fig. 1. Hydrolysis of DGalDG, MGalDG and SQDG by GPLRP2 or RPLRP2. (a) Two m g / m l DGalDG were incubated with 0.5 I x g / m l GPLRP2 at the bile salt concentrations indicated. (b) Two m g / m l DGalDG were incubated with 5 I x g / m l RPLRP2 at the bile salt concen- trations indicated. (c) Two m g / m l MGalDG and SQDG were incubated with 2 ~ g / m l GPLRP2 with 6.4 mM bile salts under conditions given in Section 2. (d) Two m g / m l MGalDG and SQDG were incubated with 2 ~ g / m l RPLRP2 with 3.2 mM bile salts under conditions given in Section 2. All results are presented as I x m o l / m i n per mg of enzyme and are means ___ S.E. of three observations.

H y d r o l y s i s o f D G a l D G b y G P L R P 2 at d i f f e r e n t b i l e s a l t c o n c e n t r a t i o n s .

350 ]" u 0 mM J T . • 3 , 2 ~

(a) 3 ° ° 1 [ / • 4,smU [r L ~ • 6,4ram

250111F I \ ~ S,0mM

~ 20o

~ 150" e

50-

0 0 5 1 0 1 5 2 0 2 5 3 0 3 5

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H y d r o l y s i s o f D G a l D G b y R P L R P 2 at d i f f e r e n t b i l e s a l t c o n c e n t r a t i o n s .

(b ) 1,2' 1 , 1 N 0 mM

• 1,6 m M 1,o A 3,2 mm 0,9- • 4,8 mM

6,4 mM 0,8 ----/k--- 9,6 mM 0,7 •

0,6" ~ O,5-

"~ 0,4-

0,3 ~

~ o,2- o,1- o,o . . . . . . . . , . . . . , . . . . , . . . . , . . . . , . . . .

0 2 0 4 0 6 0 8 0 1 0 0 1 2 0 1 4 0

T i m e ra in

H y d r o l y s i s o f M G a l D G a n d S G a l D G b y GPLRP2.

(C) 225" ---- MGalDG

200"

175

150"

~ 125"

.~ 100"

75"

o 50-

:~ 25"

• , • , • , • , • , • , .

0 5 1 0 1 5 2 0 2 5 3 0 3 5 T i m e r a i n

H y d r o l y s i s o f M G a l D G a n d S Q D G b y RPLRP2. 2 0 -

(d) • mGan~ ~ SQDG 15-!

1o

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0 1 0 2'0 3 0 4 0

T i m e ra in

L. Andersson et al. / Biochimica et Biophysica Acta 1302 (1996) 236-240 239

no l / water/ ammonia 70:20:2:1), activity of non-polar lipids migrated as FFA whereas separation of the non-polar lipids (mobile phase petroleum ether / diethylether/ acetic acid/methanol 80:20:1:2) indicated that some radioactivity also migrated as mono- and diglycerides (5% and 8%, respectively; data not shown). We therefore believe some radioactivity to be present in the hydroxy fatty acids rather than as partial diglycerides [16]. The galactose- and fatty acid analysis of the polar lipids formed from DGalDG and MGalDG, incubated with GPLRP2 (Fig. 2b, c), showed

o

o

Hydrolysis of 3H-galactose labelled DGalDG by GPLRP2.

100

90'

~0"

70"

60"

50

40'

30"

20"

10"

0 0

• ~ m ~ (a) .It DGalMG

Watersduladle

20 40 60 80 100 120 140

Time rain

Hydrolysis of 3H-fatty acid labelled DGalDG by GPLRP2,

1®t 9o: (b) l

70"

411

311

lO

0 0 2 5 20 120

Time miR

Hydrolysis of 3H-fatty acid labelled MGalDG by GPLRP2.

i • MGIIDG

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10

0 0 2 5 20 120

Time m i l

Table 1 Comparison of lipolytic activity of GPLRP2, RPLRP2, CEL, HPL and RPL on different substrates

TG Phosphatidyl- DGalDG MGalDG SQDG (tributyrin) choline

GPLRP2 2000 " 570 a 250 160 160 RPLRP2 2100 d 340 a 1 10 15 CEL 84 b 197 c 0.1 1.6 2.3 HPL 10000 none 0.2 0.3 0.1 RPL 6300 d none d 0.1 0.5 not tested

All results are given as U / m g (Ixmol fatty acid released/rain per rng of enzyme). a Hjorth et al. [7]; b BP, ickberg et al. [19]; c Lombardo et al. [20];

Jennens etal. [8].

galactose/fatty acid ratios close to 0.5 and 1, indicating that they are DGalMG and MGalMG respectively. Both MGalDG and SQDG were readily hydrolyzed by GPLRP2 (Fig, 1 c), and in both cases we saw the appearance of new more polar products on TLC and the Rf value of MGalMG was about 50% of that of MGalDG. The product from SQDG was, however, not further analyzed.

Under optimal conditions the specific activity of GPLRP2 against DGalDG was higher than for MGalDG and SQDG (Table 1). The high activity of GPLRP2 on DGalDG is on the same order of magnitude as GPLRP2 specific activity on egg yolk lecithin (Table 1). The spe- cific activity of RPLRP2 against galactolipids was 10-times lower than the activity of GPLRP2 even though it has comparable activity against phospholipids. Additionally, in contrast to GPLRP2, RPLRP2 preferred MGalDG and SQDG over DGalDG (Table 1).

4. D i s c u s s i o n

This study shows that GPLRP2 and RPLRP2 hydro- lyzes galactolipids at different rates in presence of bile salts (Fig. la, b). Incubations of GPLRP2 with [3H]galac- tose- or fatty acid-labelled substrates indicated that

Fig. 2. Hydrolysis of 3H-labelled DGalDG and MGalDG by GPLRP2. (a) Two mg /ml DGalDG containing about 60 000 dpm [ 3 H]galacto~-labelled DGalDG were incubated with 2 ~ g / m l GPLRP2 under conditions given in Section 2. The bile salt concentration was 6.4 mM. The results show percent radioactivity in DGalDG, DGalMG and water-soluble com- pounds. (b) Two mg /ml DGalDG containing about 100000 dpm [3H]fatty acid-labelled DGalDG were incubated with 2 i~g/ml GPLRP2 under conditions given in Section 2. The bile salt concentration was 6.4 mM. The results show percent radioactivity in DGalDG, DGalMG and non- polar lipids. (c) Two m g / m l MGalDG containing about 50000 dpm [3H]fatty acid-labelled MGalDG were incubated with 2 Ixg/ml GPLRP2 under conditions given in Section 2. The bile salt concentration was 6.4 mM. The results show percent radioactivity in MGalDG, MGalMG and non-polar lipids. All results are means + S.E. of three observations.

240 L. Andersson et al. / Biochimica et Biophysica Acta 1302 (1996) 236-240

GPLRP2 hydrolyzes both ester bonds of DGalDG, or that an isomerization occurs which transfers the remaining acyl chain from the s n - 2 to sn -1 posit ion to be released by GPLRP2. We could not determine if there was positional specificity. In contrast, RPLRP1 and classical pancreatic lipase showed little or no hydrolytic activity against galactolipids. The data suggest that GPLRP2 and RPLRP2 have a physiological role in the digestion of galactolipids.

The increased activity of GPLRP2 compared to RPLRP2 makes teleological sense because guinea-pigs are 100% herbivores whereas rats are not, but the explanation for the activity differences must lie in structural differences be- tween the two proteins. Comparison of their amino acid sequences shows marked homology and one notable differ- ence. GPLRP2 has a deletion in an a-hel ix loop, the lid domain, that covers the active site of PL and, by analogy, of PLRP2 [7]. The lid domain influences interfacial bind- ing and may help determine substrate specificity of pancre- atic lipases [17]. These differences in structure and the differences in substrate specificity and bile salt sensitivity suggest that the PLRP2 lipase gene subfamily may be further subdivided, with GPLRP2 and RPLRP2 being rep- resentative of two branches.

The finding of galactolipase activity in GPLRP2 and RPLRP2 provides an explanation for our earlier finding of two, separate peaks of galactol ipid-hydrolyzing activity in pancreatic juice [3]. the smaller peak corresponded to CEL and the larger peak comigrated with PL. Because PL has little activity against galactolipids, it could not account for the second peak of activity in pancreatic juice. The strong amino acid sequence homology and similar size of the recently described pancreatic l ipase-related proteins makes them strong candidates for galactolipase activity. Addit ion- ally, both PLRP1 and PLRP2 have been found in pancre- atic juice from rats and humans (Ref. [18] and Lowe, M.E., unpublished data). We speculate that HPLRP2 will hydro- lyze galactolipids and explain our earlier findings with pancreatic juice [3]. Further studies with human PLRP2 are warranted.

Acknowledgements

This research was supported by grants from the Swedish Medical Research Council (No. 3969), The Albert P~hls-

son Foundation, Scotia LipidTeknik AB and the Medical Faculty, University of Lund; also by the European Com- munities in the framework of the BIOTECH programme (BIO2-CT943041), National Institutes of Health Grant DK-33487. This work was done during the tenure of an Established Investigatorship (M.E,L.) from the American Heart Association.

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