Lipoprotein Lipase – Unstable on Purpose?

Umeå University Medical Dissertations New series No 1095 • ISSN: 036-6612 • ISBN: 978-91-7264-274-4

Department of Medical Biosciences, Physiological Chemistry, Umeå University, Umeå, Sweden

Liyan Zhang

Umeå University 2007 • UMEÅ UNIVERSITY MEDICAL DISSERTATIONS New series No 1095 ISSN number: 036-6612 ISBN number: 978-91-7264-274-4

From the Department of Medical Biosciences, Physiological Chemistry University of Umeå, Umeå, Sweden

Lipoprotein Lipase – Unstable on Purpose?

AKADEMISK AVHANDLING

som med verderbörligt tillstånd av rektorsämbetet vid Umeå universitet för avläggande av doktorsexamen i medicinsk vetenskap, offentligen kommer att försvaras i sal Betula, by 6M, Umeå universitet, onsdagen den 25 april 2007, kl. 0900

Liyan Zhang

Fakultetsopponent: Professor Cecilia Holm Institutionen för experimentell medicinsk vetenskap, Lunds universitet

• Abstract Liyan Zhang, Department of Medical Biosciences, Physiological Chemistry, Umeå University, SE- 90187 Umeå, Sweden

Lipoprotein lipase (LPL) is a central enzyme in lipid metabolism. It is a non-covalent, homo- dimeric and N-glycosylated protein, which is regulated in a tissue-specific manner and is dependent on an activator protein, apolipoprotein CII. Dissociation of active LPL dimers to monomers leads to loss of activity. This was previously found to be an important event in the rapid regulation of LPL in tissues. The mechanisms involved in the processing of LPL to active dimers, as well as in LPL inactivation through monomerization, were unknown. We have investigated the folding properties of the LPL protein, in particular the requirements for LPL to attain its active quaternary structure and to remain in the native conformation. On expression of LPL in insect cells we found that most of the LPL protein was synthesized in an inactive monomeric form. By co-expression of LPL with human molecular chaperones, especially with calreticulin (CRT), the activity of LPL increased greatly, both in the cells and in the media. The effect of CRT on LPL activity was not due to increased levels of the LPL protein, but was due to an increased proportion of active dimeric LPL. Co-immunoprecipitation experiments showed direct interaction between LPL and CRT supporting the idea that this ER-based molecular chaperone supports the formation of active LPL dimers. We showed that, bis-ANS, the aromatic hydrophobic probe 1,1΄-bis(aniline)-4,4΄- bis(naphthalene)-8,8΄disulfonate, can be used to obtain specific information about the interaction of LPL with lipid substrates and with apoCII. Bis-Ans was found to be a potent inhibitor of LPL activity, but apoCII prevented the inhibition. Our results suggest that bis-Ans binds to three exposed hydrophobic sites, of which one is at or close to the binding site(s) for apoCII. In studies of the mechanisms responsible for the spontaneous inactivation of LPL, we showed that active LPL is a dynamic dimer in which the subunits rapidly exchange partners. The rapid equilibrium between dimers and monomers exists even under conditions where LPL is relatively stable. This supports the idea that the dimer is in equilibrium with dimerization-competent, possibly active monomers. This dimerization-competent intermediate was also implicated in studies of the inactivation kinetics. The inactive LPL monomer was found to have a stable, defined conformation irrespective of how it was formed. The main differences in conformation between the inactive monomer and the active dimer were located in the middle part of the LPL subunit. Experiments with bis-Ans demonstrated that more hydrophobic regions were exposed in the inactive monomer, indicating a molten globule conformation. We concluded that the middle part of the LPL subunit is most likely engaged in the formation of the active LPL dimer. The dimerization-competent LPL monomer is a hypothetical conformational state, because it has not been possible to isolate it. To study complete refolding of LPL we used fully denatured LPL and were able to demonstrate that the recovery of LPL activity was about 40% when the denaturant was diluted by a buffer containing 20% human serum and 2M NaCl. Further studies identified calcium as the component in serum that was crucial for the reactivation of LPL. The refolding of LPL was shown to involve at least two steps, of which the first one was rapid and resulted in folded, but inactive monomers. The second step, from inactive monomers to active dimers, was slow and calcium-dependent. Also inactive monomers isolated from human tissue were able to recover activity under the influence of calcium. We proposed that calcium-dependent control of LPL dimerization might be involved in the normal post-translational regulation of LPL activity. In conclusion, LPL is a relatively unstable enzyme under physiological conditions due to its non- covalent dimeric structure. The energy barrier for folding to the active dimer is high and requires the presence of calcium ions and molecular chaperones to be overcome. The dimeric arrangement is probably essential to accomplish rapid down-regulation of LPL activity according to metabolic demand, e.g. in adipose tissue on fasting.

•

To my family

• TABLE OF CONTENTS LIST OF PAPERS...... 2 ABBREVIATIONS...... 3 INTRODUCTION...... 4 Lipoprotein Lipase ...... 4 Physiological Function ...... 4 Regulation of LPL in vivo ...... 5 Molecular Structure of LPL...... 6 LPL is a member of the lipase gene family ...... 6 Wide distribution and special site for function ...... 7 Substrate specificity...... 7 Importance of N-glycosylation...... 7 Interaction with heparin ...... 8 Dimer is the active form ...... 8 Requirement for activator ...... 8 Expression of recombinant LPL ...... 9 Mammalian expression system ...... 9 Baculovirus expression system ...... 9 Quality Control Mechanisms for proteins in the Endoplasmic Reticulum...... 10 Function of molecular chaperones...... 10 Calcium and ER molecular chaperones...... 10 AIMS OF THE PRESENT STUDY ...... 11 RESULTS AND DISCUSSION ...... 12 Paper I ...... 12 The interaction of molecular chaperones with LPL in insect cells ...... 12 Molecular chaperone(s) are directly involved in dimerization of LPL...... 12 Paper II...... 13 The fluorescent probe bis-ANS as a tool to study LPL...... 13 Functionally important hydrophobic areas on LPL ...... 13 New insight about the interaction between LPL and apoCII...... 13 On the specificity of bis-ANS for binding sites on LPL...... 14 Paper III...... 14 The explanation for the instability of LPL...... 14 Some properties of the inactive monomer - the preferred conformational state of LPL ...... 15 Paper IV ...... 15 Inactive LPL can regain its activity in vitro ...... 15 Calcium-assisted refolding pathway for LPL...... 15 What calcium-assisted refolding of LPL in vitro tells us about the potential role of calcium for the regulation of LPL activity in vivo ...... 16 General Discussion ...... 17 CONCLUSIONS...... 19 ACKNOWLEDGEMENTS ...... 20 REFERENCES...... 22

LIST OF PAPERS

This thesis is based on the four papers listed below. They will be referred to in the text by their roman numerals.

I. Calreticulin promotes folding/dimerization of lipoprotein lipase expressed in insect cells (sf21)

Liyan Zhang, Gengshu Wu, Christopher G.Tate, Aivar Lookene, and Gunilla Olivecrona. Journal of Biological Chemistry (2003) 278: 29344-51

II. 1,1'-Bis(anilino)-4-,4'-bis(naphtalene)-8,8'-disulfonate acts as an Inhibitor of Lipoprotein Lipase and Competes for Binding with Apolipoprotein CII • Aivar Lookene, Liyan Zhang , Vello Tougu, and Gunilla Olivecrona Journal of Biological Chemistry (2003) 278: 37183-94

III. Rapid subunit exchange in dimeric lipoprotein lipase and properties of the inactive monomer

Aivar Lookene, Liyan Zhang, Magnus Hultin, and Gunilla Olivecrona Journal of Biological Chemistry (2004) 279: 49964-72

IV. Calcium triggers dimerization and activation of inactive, monomeric lipoprotein lipase

Liyan Zhang, Aivar Lookene, Gengshu Wu, and Gunilla Olivecrona Journal of Biological Chemistry (2005) 280: 42580-91

2 ABBREVIATIONS

apoCII apolipoproteinCII

Bis-Ans 1,1`-Bis(aniline)-4,4`-bis(naphthalene)-8,8`-disulfonate

CRT calreticulin

ER endoplasmic reticulum

FFA free fatty acids

GdmCl guanidinium chloride

HSPG heparan-sulphate proteoglycan

kDa kilodalton

VLDL very low density lipoprotein

LPL lipoprotein lipase

2- MG 2-monoacylglycerol

3 INTRODUCTION Lipoprotein Lipase Physiological Function Lipoprotein lipase (LPL) is a glycoprotein mainly produced by parenchymal cells of adipose tissue, heart and skeletal muscle (1;2). LPL is also produced in kidney (3), lung (4), placenta (5), brain (6) as well as in pancreatic islets (7) and in macrophages (8).

Catalytically active LPL is a dimer of two identical 55 kDa subunits arranged in a head to tail fashion by non-covalent interaction (9). LPL is transported from the producing cells to the physiological site of action. This is at the endothelial surface of blood vessels, where LPL is anchored to heparan sulphate proteoglycans (HSPG) (2;10;11).

LPL hydrolyses mainly triglycerides, but also some phospholipids in triglyceride-rich lipoproteins (2;11). One source of substrate lipoproteins is dietary fat (exogenous lipids), which are assembled into chylomicrons in the intestinal mucosa. Another source is endogenous lipids, which are assembled into very low density lipoproteins (VLDL) in the liver. After entry into the blood, chylomicrons are hydrolysed by LPL at the endothelial cell surface. Chylomicron remnants, containing apoB-48 and apoE, are taken up by the • liver via the chylomicron remnant receptors recognizing apoE (12). Lipids taken up by the liver from chylomicron remnants may be subsequently utilized for synthesis of very low density lipoproteins. VLDL encounters LPL at the vascular endothelium in the same way as chylomicrons. As a result of hydrolysis, VLDL is reduced in size to intermediate density lipoprotein remnants that are further remodelled into low density lipoproteins (LDL) and high density lipoproteins (HDL). The hydrolysis of triglycerides present in circulating chylomicrons and VLDL provides non-esterified fatty acids and 2-monoacylglycerol for tissue utilization (2;11).

In addition, LPL can bind simultaneously to lipoproteins and to specific cell surface receptors, such as the LDL receptor-related protein (13), and HSPG (14;15). The presence of LPL leads to increased uptake of lipoproteins or lipoprotein constituents into cells by receptor-mediated endocytosis. The physiological function of LPL thus includes both the catalytic function with regard to hydrolysis of lipids in plasma, and the bridging function with regard to LPL mediated transfer of lipoprotein constituents to cells without hydrolysis of the lipids.

4 Figure 1 The dual function of LPL; as an enzyme and as a ligand for binding of lipoproteins to cell surfaces via HSPG and/or receptors

Regulation of LPL in vivo LPL is regulated by various factors, such as nutrition, hormones, pathological and physiological environment, at the transcriptional, translational as well as post-translational level (2;11;16).

The regulation of LPL activity is tissue-specific. LPL activity in white adipose tissue is inversely modulated compared to LPL in heart and skeletal muscle in several situations. LPL activity in adipose tissue from fed rats is higher compared to fasted rats (17-20), whereas the opposite trend is found in the heart and skeletal muscles, especially when the extracellular pool of LPL in these tissues is studied (21-23). During long- term exercise, LPL activity in skeletal muscle increases whereas the activity in adipose tissue and heart is stable (24). Insulin increases LPL activity in adipose tissue but decreases LPL activity in muscle during short-term insulin infusion (25). Epinephrine reduces LPL activity in adipose tissue but stimulates LPL activity in skeletal muscle (26).

The tissue-specific regulation of LPL activity implies that different mechanisms for control of LPL operate in the respective tissues. Recent studies have shown that, during short-term fasting, the modulation of LPL activity occurs in the extracellular, endothelial- bound part not only in adipose tissue (19;20) but also in heart and skeletal muscle (27). Regulation involves a shift of LPL from active dimers to inactive monomers in adipose tissue when going from fed to fasted conditions (28). This shift was shown to be dependent on expression of another gene, “LPL-controlling protein”, which is turned over rapidly (19). Angiopoietin-like protein 4 was found recently to be the fasting-induced controller of LPL in adipose tissue (29). A similar process appears to control LPL activity in heart and

5 skeletal muscle, but in the opposite direction, i.e. high in the fasted state and low in the fed state (27).

Molecular Structure of LPL The 3-D structure of the LPL subunit, as shown in figure 2, has been modelled based on the X-ray structure of pancreatic lipase (30;31), a lipase from the same gene family with high sequence homology to LPL. According to this model, LPL consists of two structurally distinct regions (30;32), a larger N-terminal domain (residues 1-312 based on human LPL), which forms an α/β-structure, and a smaller C-terminal domain (residues 313-448), which is rich in antiparallel β sheets (28). The N-terminal domain with the catalytic traid (Ser132, Asp156 and His241) is covered by a lid, whereas the C-terminal domain is required for binding of lipids (32;33), lipoprotein receptors (12;34) and HSPG (35;36).

The structure of LPL is stabilized by disulfide bridges (37).There are four cysteine pairs that are completely conserved among LPL of all species known. Three of them (Cys216- Cys239, Cys264-Cys275 and Cys278-Cys283) stabilize the folding of the N-terminal domain, and are critical for the catalytic function of LPL, whereas the fourth (Cys418-Cys438) is in the C-terminal domain and it is not crucial • for the catalytic function (31;37).

There are three consensus sites (Asn43, Asn257, and Asn359) for N-linked glycosylation in LPL (38-41). Guinea pig and chicken LPL have three oligosaccharide chains (two complex and one high mannose) (42;43), but LPL from other species including humans have only two oligosaccharide chains (44;45), since Asn257 is not glycosylated. Figure 2 Model of the LPL structure

LPL is a member of the lipase gene family LPL, pancreatic lipase, hepatic lipase (46), endothelial lipase (47) and phosphatidylserine phospholipase A1 (48), as well as the most recently discovered lipase H (49), belong to the same gene family but play different roles in lipid metabolism (46;50). Considering the high degree of structural similarity between the lipase family members, one could ask what are the special properties of LPL.

6 Wide distribution and special site for function Differently from the other members of the lipase family, LPL is widely distributed in various tissues and cells. Pancreatic lipase is synthesized by exocrine pancreatic cells and is secreted into the intestinal lumen (51). Hepatic lipase is synthesized exclusively in the liver and in the adrenal glands (52). Endothelial lipase is expressed by endothelial cells (47), but also elsewhere, primarily in the placenta (53) and lung. Phosphatidylserine phospholipase A1 is produced exclusively by platelets (48), whereas lipase H is found mainly in the intestine (49). The physiological lipid substrate for lipase H has not been identified and little is known about its function.

Unlike some of the other family members, LPL has to be transported from its places of synthesis to the endothelium to perform its functions, such as from adipocytes to endothelial cells, whereas hepatic lipase and endothelial lipase act on the surface of the cells in which they are produced.

Substrate specificity LPL preferably catalyzes the hydrolysis of triglycerides, but phospholipids are hydrolyzed by the enzyme with lower rates (2). Other lipases in the lipase gene family show higher phospholipase activity, such as hepatic lipase (54), endothelial lipase (55) and even more true for phosphatidylserine phospholipase A1 (48). When comparing LPL and hepatic lipase, it was concluded that the main determination for substrate specificity was the lid structure.

Importance of N-glycosylation N-glycosylation is a prerequisite for LPL to obtain catalytic activity. Among the rest of the lipases, hepatic lipase and endothelial lipase have been shown to be dependent on the N- terminal glycan chain for activity (44). Pancreatic lipase displays full enzymatic activity even when expressed in lower eukaryotic cells, such as insect (56) and yeast (57) cells. For LPL, even when expressed in mammalian cells, only part of the newly synthesized LPL protein acquires native conformation. The remainder of the LPL protein ends up in misfolded aggregates that are degraded in the ER (41). Pancreatic lipase is not affected by the murine combined lipase deficiency (cld/cld) mutation, which virtually abolishes the activity of LPL and hepatic lipase (58;59). Thus, pancreatic lipase must be able to acquire full catalytic activity with little assistance from chaperones or glycosylation.

7 Interaction with heparin There are several clusters of basic amino acids that were demonstrated to represent heparin-binding domains on LPL (60). Similar to LPL, hepatic lipase binds to heparin, but with lower affinity (2). The putative heparin-binding sites present in LPL are highly conserved in endothelial lipase, but it is unclear if the binding affinity to heparin of endothelial lipase is lower or comparable to that of LPL (61;62).

Dimer is the active form Catalytically active LPL is a homodimer (9;63). Hepatic lipase has also been demonstrated to be a dimer (64). In contrast, pancreatic lipase is active as a monomer, while other lipases need further investigation regarding their active state. The nature of intermolecular interactions between two subunits of LPL are suggested to be of electrostatic as well as of hydrophobic character (65). The sites on LPL that are involved in dimerization have not been identified.

Requirement for activator LPL has basal activity towards some substrates (66). For hydrolysis of substrates containing fatty acyl chains longer than seven carbons, LPL needs an activator, • apolipoprotein CII (apoCII), for full activity (2). ApoCII activation is unique to LPL since the activity of other lipases is not increased by apolipoproteins although pancreatic lipase also needs activator, i.e. colipase (67). ApoCII and colipase are structurally very different and probably fulfil different functions. Colipase anchors pancreatic lipase on the lipid surface and protects it from being dissociated from the lipid droplets by bile salts (51), but it does not influence the rate of hydrolysis. LPL binds well to lipid even in the absence of apoCII (66).

ApoCII, a 79-amino acid residue peptide, belongs to the exchangeable apolipoproteins on the surface of lipoproteins. Studies with 2D NMR have shown that apoCII contains three regions with amphipathic helical conformation (68). The LPL-activating region of apoCII has been localized to the C-terminal third helix (69;70), whereas the N-terminal two-thirds of the molecule are involved in lipid binding.

It is believed that the basis to activate LPL by apoCII is the formation of a complex between apoCII and LPL at the surface of a lipid droplet (70). The detailed mechanism for the activation remains unclear.

8 The site on LPL that interacts with apoCII has not been finally defined. It was suggested that the binding sites for apoCII may be either across the anti-parallel LPL dimer with binding sites both on the N-terminal and C-terminal folding domains of LPL (71). Other investigations suggested that binding occurs at the interface between the N-terminal and C- terminal folding domains of LPL and also engage the lid region that covers the active site, in close proximity to the active site pocket (72). McIlhargey et al speculated, based on cross-linking experiments and mutagensis, that the main interaction between LPL and apoCII must be electrostatic (71).

Expression of recombinant LPL Mammalian expression system LPL and many LPL mutants have been transiently expressed in COS cells, CHO-cells or HEK-cells by several research groups (73;74). This has allowed studies on different aspects of LPL function, e.g. structural requirements for activity, substrate preference and binding to heparin, to lipoproteins and to receptors. However, in these systems the expression level of LPL is low (around 0.5µg/ml) and large-scale production of LPL protein for direct studies by biophysical techniques is not suitable. The requirement for serum in the culture media is another disadvantage, usually preventing complete purification of the recombinant LPL.

Baculovirus expression system This system for production of recombinant proteins by using insect cell lines, such as SF9, SF21 or high five is well established (75). Insect cells can perform most of the post- translational processing steps for proteins occurring in mammalian cells (76). With respect to the ability to produce fully processed and biologically active recombinant proteins, insect cells are considered to be the second choice after mammalian cells (77). The advantage of the baculovirus system over the mammalian cell systems is that the insect cells can be grown in large scale in suspension cultures at a relatively low cost. The use of serum free growth media simplifies the purification of the recombinant product (77). The baculovirus system has been successfully used for production of pancreatic lipase, enabling crystallography of the lipase and its complex with colipase (78). This system has also been used for production of hormone-sensitive lipase (79).

9 Quality Control Mechanisms for proteins in the Endoplasmic Reticulum Function of molecular chaperones The quality-control mechanism, that prevents newly synthesized, misfolded proteins from being transported further in the cells, is dependent on N-glycosylation of the proteins (80;81). The role of calnexin/calreticulin in the endoplasmic reticulum (ER) is to increase the folding efficiency of glycoproteins, and to prevent premature oligomeric assembly (aggregation) (82). They are examples of molecular chaperones. As shown in Fig 3, oligosaccharide chains are added to the newly synthesized proteins. The glucose units on the newly synthesized glycoprotein are trimmed by glucosidase I and II to form a monoglucosylated protein which is subsequently recognized by calnexin/calreticulin (Fig 2). Interaction of the glycoprotein with calnexin/calreticulin promotes interaction with the

co-chaperone ERp57, a thiol disulfide oxidoreductase, which interacts with the p-domain of calnexin/calreticulin as well as with the cysteines in the glycoprotein (83). When the glycoprotein is released from the chaperones, it is examined by glucosyl transferase, a folding sensor in ER, which allows only correctly folded and assembled proteins to be transported from ER to Golgi, while insufficiently folded molecules enter the • calnexin/calreticulin cycle again (83;84).

Calcium and ER molecular chaperones Calreticulin is a Ca2+-binding chaperone. It binds Ca2+ with high capacity in the ER lumen and is thereby able to influence the folding and post-translational modification of virtually all glycosylated, secreted, or integral membrane proteins that pass through the ER (85). Binding of calreticulin to newly synthesized glycoproteins is sensitive to the Ca2+ concentration, suggesting that changes in the level of ER luminal Ca2+ may affect the folding of glycoproteins (85;86).

Figure 3 Schematic view the interaction of glycoproteins with ER chaperones

AIMS OF THE PRESENT STUDY

• To establish an expression system that can produce LPL for structural studies with biophysical methods • To investigate the role of molecular chaperones for folding/dimerization of LPL • To localize the binding region in LPL for apoCII • To study the mechanisms responsible for the spontaneous inactivation of LPL • To study the properties of inactive LPL monomers • To explore possible refolding pathways for denatured LPL to the native state in vitro

11 RESULTS AND DISCUSSION Paper I The interaction of molecular chaperones with LPL in insect cells Formation of active LPL requires glycosylation and dimerization of LPL subunits in the ER. To get sufficient amounts of active LPL for structural studies, we decided to express recombinant human LPL by the baculovirus system. We found that the insect cells produced and secreted LPL with proper glycosylation pattern, but most of the LPL protein was inactive. Based on the role of ER molecular chaperones in the folding of proteins, we speculated that insect cells might lack the molecular chaperones that are needed to promote intracellular maturation of LPL in mammalian cells. Therefore, we selected a number of folding-assistant factors, including calnexin, calreticulin (CRT), protein disulfide

isomerase, immunoglobulin heavy chain binding protein, and the foldase ERp57 for co- expression with LPL. We found that calnexin and especially (CRT) increased LPL activity in the baculovirus system.

Molecular chaperone(s) are directly involved in dimerization of LPL To elucidate the mechanism, by which the LPL activity was enhanced, the effect of co- • expression of LPL with CRT was further studied. Analysis by heparin-sepharose chromatography and sucrose density gradient centrifugation revealed that the active LPL produced by the insect cells was a dimer with high affinity to heparin. Co-expression with CRT increased the proportion of active LPL from less than 10% of the total LPL protein to about 50%, without significant change of the amount of total LPL protein mass. Thus, CRT had a remarkable effect on LPL specific activity. This was true both in the media and in the cells indicating that CRT did not primarily affect the secretion of LPL. However, when castanospermine, an inhibitor of glucosidase I or II was used together with CRT, a decrease in LPL specific activity was observed, indicating the dependence of LPL activity on interaction with CRT. Immunoprecipitation of cell extracts showed that CRT was immuno-precipitated by chicken immunoglobulin to LPL when CRT were co-expressed with LPL, but not when CRT was expressed alone. This demonstrated that CRT interacted with LPL to support the proper folding of newly synthesized LPL protein, and thereby stimulate dimerization of LPL in the ER.

12 Paper II The fluorescent probe bis-ANS as a tool to study LPL The hydrophobic fluorescent probe 1,1'-bis(anilino)-4,4'-bis(naphthalene)-8,8'-disulfonate (bis-ANS) is often used as a sensitive probe to detect hydrophobic sites and conformational changes in proteins. An additional advantage with this compound is the possibility to study irreversible binding of the probe after photoincorporation by irradiation with UV light. The preliminary aim was to use bis-ANS for these purposes in studies of LPL. We found, however, that bis-ANS is a potent inhibitor of LPL, and that bis-ANS binds to LPL with very high affinity compared to other proteins. Two important interactions of LPL, with apoCII and with individual substrate molecules, were affected by the binding of bis-ANS. Therefore bis-ANS can be used to obtain specific information with regard to hydrophobic regions in LPL, as well as the interaction of LPL with lipid substrates and with apoCII.

Functionally important hydrophobic areas on LPL Studies with bis-ANS, by fluorescence titration and surface plasmon resonance, revealed that there may be at least three largely independent but functionally important hydrophobic sites on LPL; one for interfacial binding, one for binding of fatty acids, and one for binding of apoCII and possibly also for individual substrate molecules. This third site may be the most specific, since it appears to be present only on correctly folded dimers of LPL, and it was not present in other lipases. Our data indicated that this region is exposed in water solution, because the binding of bis-ANS to LPL was seen in buffer in the absence of a lipid/water interface. Most likely this site does not involve the active site lid, since bis- ANS did not disturb the very sensitive trypsin cleavage site at the tip of the lid.

New insight about the interaction between LPL and apoCII The most important observation in this study was that the inhibitory effect of bis-ANS on LPL activity could be abolished by addition of apoCII. The mechanism for this is most likely the displacement of bis-ANS from LPL through competition for the same site by apoCII. We found that the amount of apoCII needed to restore LPL activity increased with increasing concentrations of bis-ANS, indicating that apoCII and bis-ANS competed for the same sites. Binding of bis-ANS to LPL did not cause dissociation of the LPL dimer into inactive monomers and bis-ANS did not hamper the interfacial binding of LPL to lipid emulsions, but the compound affected directly the catalytic activity of LPL.

13 On the specificity of bis-ANS for binding sites on LPL We studied whether bis-ANS could bind to and inhibit other lipases, such as pancreatic lipase. This enzyme is similar to LPL both in structure and in catalytic function. The result showed that pancreatic lipase was not inhibited even at very high concentrations of bis- ANS. Apart from this, we also found that only the active, wild-type form of human apoCII was able to restore the inhibited activity by bis-ANS, whereas mutants, with one residue exchanged in the putative binding site for LPL, were completely inactive. In addition, the stoichiometry of the binding of bis-ANS to LPL indicated that, despite the possibility that several bis-ANS molecules might bind per LPL dimer, there were only one or two high affinity binding sites on dimeric LPL. Inactive monomeric LPL did not bind bis-ANS with high affinity, demonstrating that the high affinity sites were somehow connected to the active conformation of LPL. We concluded that bis-ANS probably binds in a specialized, exposed hydrophobic region close to the active site and that this region may be the binding site for apoCII.

Paper III The explanation for the instability of LPL • Many previous studies in vitro have shown that the active dimeric state of LPL is unstable. Osborne et al had shown that the inactivation rate is dependent on the lipase protein concentration, and that inactivation of LPL includes a step of reversible dissociation of the dimer followed by a step of irreversible unfolding of the dissociated monomers (63). To further explore this mechanism, we used different techniques, such as fluorescence energy transfer, streptavidin-agarose chromatography and compartmental modelling of the inactivation kinetics. We demonstrated that active LPL is a dynamic dimer in which the subunits rapidly change partners. The rapid exchange occurs even under conditions when LPL is relatively stable, such as at low temperature, and in the presence of either high concentration of NaCl or heparin. These data supported the hypothesis that LPL dimers are in equilibrium with dimerization-competent monomers. However, we can’t deduce with certainty whether this intermediate is active or inactive since it has not been possible to isolate it. We assume that LPL in this state may either associate reversibly to active dimers/oligomers, or go through conformational changes to an inactive, misfolded state of the monomer. This is most likely equivalent to the inactive form of LPL found in tissues in vivo (28).

14 Some properties of the inactive monomer - the preferred conformational state of LPL Inactive, monomeric LPL has a strong tendency to aggregate. The aggregation was much influenced by temperature, concentration of NaCl and other stabilizers such as lipoproteins, albumin and heparin. We demonstrated, by binding of LPL to bis-ANS, that hydrophobic areas are more exposed in the LPL monomer than in the dimer. This may explain the strong propensity of monomers to aggregate. However, aggregation occurred slowly in the presence of high concentration of NaCl which indicates that ionic interaction may play a greater role for aggregation than hydrophobic interactions.

Limited proteolysis of monomeric LPL with proteinases demonstrated that the monomer was more susceptible to cleavage than the LPL dimer. With endo-Glu-C, three peptides were generated from LPL monomer, whereas dimeric LPL was totally resistant to cleavage even after treatment for 24 h. The initial cleavage fragment obtained from the LPL monomer was shown to be the N-terminal part of the N-terminal domain.

Paper IV Inactive LPL can regain its activity in vitro We speculated that a refolding pathway could exist from unfolded or misfolded monomers, and that studies on LPL refolding in vitro would provide important information about the control of LPL dimerization at the molecular level in vivo. To investigate refolding we used purified bovine LPL which was fully denatured in 6 M guanidinium – hydrochloride (GdmCl). We found that it was possible to regain about 40% of the catalytic activity of LPL after dilution to non-denaturing concentrations of GdmCl in a medium containing human serum. This was a remarkable finding since inactivation of LPL was previously considered to be totally irreversible. Subsequently, we identified calcium as the crucial component in serum for reactivation of LPL.

Calcium-assisted refolding pathway for LPL We discovered that refolding of fully denatured LPL involved two steps. For the first step we used 2 M NaCl and VLDL to prevent aggregation of the intermediate, inactive form of LPL. Measurement of far-UV circular dichroism and fluorescence showed that under these conditions LPL was in the “molten globule”, loosely folded state. Sucrose gradient ultracentrifugation and heparin affinity chromatography further demonstrated that LPL in this state was inactive monomer. Calcium was not needed for this first step. In the second step, calcium ions probably interacted with the non-native LPL and triggered conformational changes to the active dimeric state. Sucrose gradient ultracentrifugation and

15 chromatography on heparin-Sepharose demonstrated that the reactivated LPL was a dimer with high affinity for heparin.

The refolding process in the second step was slow, temperature-dependent, and also highly dependent on other conditions, indicating that an energy barrier had to be overcome. Proline isomerisation rather than the actual dimerization was most likely the rate-limiting step for the complete refolding of LPL.

What calcium-assisted refolding of LPL in vitro tells us about the potential role of calcium for the regulation of LPL activity in vivo In the ER lumen, Ca2+ is bound to resident chaperones, but Ca2+ also plays a dynamic role for other aspects of ER function (87). In view of the recovery of LPL activity in the presence of calreticulin and Ca2+ as well as the strong relationship between Ca2+ and calreticulin function, it is tempting to speculate that Ca2+ may be involved in the regulation of LPL activity in vivo.

Rapid modulation of LPL activity by Ca2+ has previously been reported from cell culture experiments where recovery of LPL activity in adipocytes was proportional to the 2+ • concentration of Ca (88). It was also demonstrated, in CHO-K1 cells (Chinese hamster ovary K1 cells), that ER Ca2+ depletion, either by stimulation of IP3-generation or by exhaustive Ca2+ depletion by a sarcoplasmic/ER Ca2+-ATPase agonist, significantly attenuated LPL activity (86). In paper I, we demonstrated that over-expression of calreticulin in the insect cells promoted dimerization of LPL. In paper IV, we demonstrated that the calcium-assisted refolding of LPL resembles the two-step binding/release mechanism of the chaperone system in the ER. It is known that over-expression of calreticulin leads to increased amounts of Ca2+ in intracellular stores (85). Thus, folding of LPL in the ER might be assisted both by chaperones and by Ca2+ in vivo. There is as yet no study in vivo of the relation between changes of cytosolic Ca2+ and LPL activity, but it is known that alterations of cytosolic Ca2+ are related to decreased storage of Ca2+ in ER (87). LPL activity is rapidly modulated in skeletal muscle in response to physical activity. This is in line with the role of Ca2+ for controlling contraction. Intracellular Ca2+ homeostasis could be an early target of cell dysfunction in metabolic diseases, such as in diabetes mellitus or atherosclerosis (89;90). An interesting possibility is that disease-associated alterations in intracellular Ca2+ may influence the regulation of LPL activity.

16 General Discussion The action of LPL was discovered more 60 years ago (91), but the regulation of LPL activity is still a hot topic. Everyday regulation occurs on the post-translational level and involves dissociation of active LPL into monomers mediated by other gene products acting as LPL controllers (19;20;29).

What is the molecular mechanism involved in the inactivation of LPL? Our new understanding is that the dynamic interaction of the subunits in the LPL dimer is crucial. The association between LPL and heparin or HSPG is characterized by fast kinetics with high rate constants for both binding and dissociation of LPL to heparin/HSPG resulting in a rapid movement of LPL molecules between its binding sites on heparin/HSPG (92). Considering that the off-rate for binding of LPL to heparin/HSPG and to lipoproteins is faster than the rate for LPL subunit exchange, it is suggested that the well-known effect of heparin and of lipoproteins on LPL stability is to stabilize the dissociated monomer rather than to keep the dimer together. For regulation of LPL at the endothelial binding sites in vivo, we believe that, during the process of subunit exchange, factors, such as the recently recognized angiopoietin-like protein 4 which is upregulated in adipose tissue on fasting, binds to the transiently exposed monomers (29). This may interfere with the ability of HSPG to stabilize the dimerization-competent monomer. As a consequence, conformational changes of the monomer may occur, converting the folded monomer to a misfolded monomer. The result will be a decrease in LPL activity. The nature and regulation of controlling factors equivalent to angiopoietin-like protein 4 may be tissue specific (27;29). It is important that LPL can be controlled rapidly, even when the enzyme is located extracellularly, at its endothelial binding sites. There the enzyme is out of reach of commonly used cellular control mechanisms such as phosphorylation or binding of intracellular ligands such as nucleotides and other signals of the energy status.

Recent data from Lutz et al indicated that transient dissociation of LPL dimer may be important for the intracellular transportation or secretion of LPL (93). This group constructed recombinant dimeric LPL which had the monomer subunits linked covalently, and found that this form of LPL dimers had the same stability as the non-covalent linked LPL. Mice expressing this construct on an LPL (-/-) background had lower levels of LPL protein mass and activity in postheparin plasma compared to transgenes with the normal LPL. The postheparin plasma did not contain covalently linked dimers. The reason was probably that LPL could not be secreted as a covalent dimer, and had to be clipped before

17 secretion. These experiments indicate that secretion of noncovalent dimers is favoured. Thus, the rapid dissociation/association of the subunits in LPL dimers may be important for the transport and processing of the LPL protein for secretion.

We learned, from studies in paper I, that proper folding of the monomer is a major bottleneck for synthesis of active LPL. In the studies in paper II and IV, we learned that stabilization of the LPL monomer is crucial to preserve activity. Thus, LPL is a metastable dimer that has a built-in propensity to loose activity through dissociation into monomers. This ability of LPL to be rapidly controlled by a conformational change may be an important protective property so that LPL at the endothelial binding sites can be switched off on given signals to prevent excessive uptake of lipids, and hence lipotoxicity, in organs and tissues.

dimerization momerization •

Newly synthesized monomer Active dimer Dissociated monomer

Figure 4 Process of dimerization and monomerization of LPL

CONCLUSIONS

• Co-expression of LPL with human calreticulin in insect cells increased the

specific activity of the LPL protein, demonstrating that calreticulin supports

folding and dimerization of LPL

• Bis-ANS is a potent inhibitor of LPL and it binds to an exposed hydrophobic

area on LPL at or near the binding site(s) for apoCII and for individual lipid

substrate molecules.

• Active LPL is a dynamic dimer in which the subunits rapidly exchange

partners.

• The largest conformational change on dissociation of the LPL dimer is in the

middle of the LPL subunit. This part is probably engaged in subunit interaction

in the active dimer. Fully denatured LPL can be reactivated by dilution in

serum. Calcium was found to be the crucial component in serum for

reactivation.

• Calcium-assisted folding of LPL from the fully denatured state involves at least

two steps. The first step is rapid and calcium-independent, while the second

step is a calcium–dependent and slow process. Proline isomerisation appears to

be rate-limiting for this step

19 ACKNOWLEDGEMENTS

The present study has been performed at the Department of Medical Biosciences, Physiological Chemistry, Umeå University, Sweden. I would like to express my sincere gratitude and best wishes to: Gunilla Olivecrona: My supervisor, thank you not only for the scientific training and advice, but also for every personal help! You are always in my thoughts. Thomas Olivecrona: My “unofficial supervisor”, thank you for all the personal help and support in science, for being so friendly and helpful on checking my thesis. You are always a great character in my heart. Wishing you many sunny days all the way to the Golf course! Aivar Lookene: My co-supervisor, thank you for all the valuable discussion and fruitful collaboration. I really enjoy working with you. Wish you every success in life! Terry Persson: It is really a blessing to have you beside me during these years. You make my world glow with so much relief, thank you for everything! Kerstin Jonhasson: I am lucky to have a wonderful friend like you. It does not matter wherever I am, I will always miss you. Solveig Nilsson: Always considerate and fill my • working days with your tea! Lotta and Åsa: People often say that life is a beautiful journey, I say, yes, especially when one has someone like you with a smile to share everyday. Elena: For all the rides to different activities. Olessia: For all the joy we shared, thanks for being there by my side; Valya: Know you’re coming by the sound of your fast footsteps; will remember you as being a hard worker. Lars Bläckberg: For all the suggestions whenever I need advice, also for being so helpful and friendly in correcting things from my thesis. Stefan Nilsson: Thank you for sharing your time with me on Biacore. Lars Frängsmyr: For all the chats, for the stories and jokes, I like them, especially the one when you mimic Aivar looking at the Biacore screen. Whenever I enter the small Biacore room, I can’t help laughing (even now when I am writing this). Emma Farlgren: Thank you so much for all the hands you gave me, good luck with your new study! Michael Edlund: For being my teacher working with baculovirus system; Ludmilla Morozova-Roche: for being warm and considerate; for trying to drive away my melody from “blues” to “sweet”. The pediatric girls: Susanne Lindqvist, Yvonne Andersson, Carina Lagerqvist, Anna Möllsten: Nice to get to know you and best wishes to all of you! Thank you to everyone at the Department of Medical Biosciences who all contribute to creating a pleasant working environment.

20 All my Chinese friends: Yan Shen, HuXiaolei and Li Xiaonan, speaking the native language is part of our joy, discussing other issues was also fun. I cherish every moment we shared! Li Donghou and Jin Fusan: I miss you guys dearly! You have made my life different; I feel that I am never that far from you! Luo Yang: For your precious friendship and support. A smile comes to my face whenever I recall the time we spend with each other. Best wishes to you! Zhai Ning: What can I say about such a cool and clever friend as you! Wish you love, luck and laughter! Lu Zhiyu and Lijin: You are both always on my mind! Life was full of music when we were together. I still remember the scene we made with the tooth paste climbing the stairs… It was really fun and I will always treasure this moment! Hope you are surrounded by beautiful days every day! My relatives in the big family: Someone says thank you with a gift, someone says it with a hug, but I say it with my heart, a heart with endless love! Yang: My lovely boy! It is a real gift and blessing to have such a wonderful son as you, I thank God for this! You make every moment of our life special. Wish you happiness everyday! Gengshu: My beloved husband, the one who cares, the one who shares, and the one who gives me a shoulder to lean when I am tired, thank you for your endless love and faithful support! May good fortune always shine on you!

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