Identification of a Lipoprotein Lipase Cofactor-binding Site by Chemical Cross-linking and Transfer of Apolipoprotein C-II-responsive Lipolysis from Lipoprotein Lipase to Hepatic Lipase
2003; Elsevier BV; Volume: 278; Issue: 25 Linguagem: Inglês
10.1074/jbc.m300315200
ISSN1083-351X
AutoresTrina Leann McIlhargey, Yingying Yang, Howard Wong, John S. Hill,
Tópico(s)Lipid metabolism and disorders
ResumoTo localize the regions of lipoprotein lipase (LPL) that are responsive to activation by apoC-II, an apoC-II peptide fragment was cross-linked to bovine LPL. Following chemical hydrolysis and peptide separation, a specific fragment of LPL (residues 65–86) was identified to interact with apoC-II. The fragment contains regions of amino acid sequence dissimilarity compared with hepatic lipase (HL), a member of the same gene family that is not responsive to apoC-II. Using site-directed mutagenesis, two sets of chimeras were created in which the two regions of human LPL (residues 65–68 and 73–79) were exchanged with the corresponding human HL sequences. The chimeras consisted of an HL backbone with the suspected LPL regions replacing the corresponding HL sequences either individually (HLLPL-(65–68) and HLLPL-(73–79)) or together (HLLPLD). Similarly, LPL chimeras were created in which the candidate regions were replaced with the corresponding HL sequences (LPLHL-(77–80), LPLHL-(85–91), and LPLHLD). Using a synthetic triolein substrate, the lipase activity of the purified enzymes was measured in the presence and absence of apoC-II. Addition of apoC-II to HLLPL-(65–68) and HLLPL-(73–79) did not significantly alter their enzyme activity. However, the activity of HLLPLD increased ∼5-fold in the presence of apoC-II compared with an increase in native LPL activity of ∼11-fold. Addition of apoC-II to LPLHL-(77–80) resulted in ∼10-fold activation, whereas only ∼6- and ∼4-fold activation of enzyme activity was observed in LPLHL-(85–91) and LPLHLD, respectively. In summary, our results have identified 11 amino acid residues in the N-terminal domain of LPL (residues 65–68 and 73–79) that appear to act cooperatively to enable substantial activation of human LPL by apoC-II. To localize the regions of lipoprotein lipase (LPL) that are responsive to activation by apoC-II, an apoC-II peptide fragment was cross-linked to bovine LPL. Following chemical hydrolysis and peptide separation, a specific fragment of LPL (residues 65–86) was identified to interact with apoC-II. The fragment contains regions of amino acid sequence dissimilarity compared with hepatic lipase (HL), a member of the same gene family that is not responsive to apoC-II. Using site-directed mutagenesis, two sets of chimeras were created in which the two regions of human LPL (residues 65–68 and 73–79) were exchanged with the corresponding human HL sequences. The chimeras consisted of an HL backbone with the suspected LPL regions replacing the corresponding HL sequences either individually (HLLPL-(65–68) and HLLPL-(73–79)) or together (HLLPLD). Similarly, LPL chimeras were created in which the candidate regions were replaced with the corresponding HL sequences (LPLHL-(77–80), LPLHL-(85–91), and LPLHLD). Using a synthetic triolein substrate, the lipase activity of the purified enzymes was measured in the presence and absence of apoC-II. Addition of apoC-II to HLLPL-(65–68) and HLLPL-(73–79) did not significantly alter their enzyme activity. However, the activity of HLLPLD increased ∼5-fold in the presence of apoC-II compared with an increase in native LPL activity of ∼11-fold. Addition of apoC-II to LPLHL-(77–80) resulted in ∼10-fold activation, whereas only ∼6- and ∼4-fold activation of enzyme activity was observed in LPLHL-(85–91) and LPLHLD, respectively. In summary, our results have identified 11 amino acid residues in the N-terminal domain of LPL (residues 65–68 and 73–79) that appear to act cooperatively to enable substantial activation of human LPL by apoC-II. Hepatic lipase (HL) 1The abbreviations used are: HL, hepatic lipase; LPL, lipoprotein lipase; SASD, sulfosuccinimidyl-2-(p-azidosalicylamido)ethyl 1,3′-dithiopropionate; PBS, phosphate-buffered saline. 1The abbreviations used are: HL, hepatic lipase; LPL, lipoprotein lipase; SASD, sulfosuccinimidyl-2-(p-azidosalicylamido)ethyl 1,3′-dithiopropionate; PBS, phosphate-buffered saline. and lipoprotein lipase (LPL) are members of the same lipase gene family, along with pancreatic lipase, the pancreatic lipase-related lipases, endothelial lipase, and phosphatidylserine-specific phospholipase A1 (1Ben-Zeev O. Ben-Avram C.M. Wong H. Nikazy J. Shively J.E. Schotz M.C. Biochim. Biophys. Acta. 1987; 919: 13-20Crossref PubMed Scopus (27) Google Scholar, 2Kirchgessner T.G. Chaut J.-C. Heinzmann C. Etienne J. Guilhot S. Svenson K. Ameis D. Pilon C. D'Auriol L. Andalibi A. Schotz M.C. Galibert F. Lusis A.J. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 9647-9651Crossref PubMed Scopus (185) Google Scholar, 3Hide W.A. Chan L. Li W.-H. J. Lipid Res. 1992; 33: 167-178Abstract Full Text PDF PubMed Google Scholar, 4Hirata K.-i. Dichek H.L. Cioffi J.A. Choi S.Y. Leeper N.J. Quintana L. Kronmal G.S. Cooper A.D. Quertermous T. J. Biol. Chem. 1999; 274: 14170-14175Abstract Full Text Full Text PDF PubMed Scopus (262) Google Scholar, 5Jaye M. Lynch K.J. Krawiec J. Marchadier D. Maugeais C. Doan K. South V. Amin D. Perrone M. Rader D.J. Nat. Genet. 1999; 21: 424-428Crossref PubMed Scopus (418) Google Scholar, 6Wong H. Schotz M.C. J. Lipid Res. 2002; 43: 993-999Abstract Full Text Full Text PDF PubMed Scopus (237) Google Scholar). Through their ability to hydrolyze triglycerides and phospholipids in a variety of circulating plasma lipoproteins, including chylomicrons and very low, intermediate, and high density lipoproteins, HL and LPL greatly influence lipid metabolism (7Semenkovich C.F. Luo C.-C. Nakanishi M.K. Chen S.-H. Smith L.C. Chan L. J. Biol. Chem. 1990; 265: 5429-5433Abstract Full Text PDF PubMed Google Scholar, 8Olivecrona T. Bengtsson-Olivecrona G. Curr. Opin. Lipidol. 1990; 1: 222-230Crossref Scopus (81) Google Scholar, 9Olivecrona T. Bengtsson-Olivecrona G. Curr. Opin. Lipidol. 1993; 4: 187-196Crossref Scopus (122) Google Scholar). HL and LPL are associated with cell surfaces through an interaction with heparan sulfate proteoglycans and are thought to possess non-catalytic functions associated with the binding and clearance of various lipoproteins (10Amar M.J. Dugi K.A. Haudenschild C.C. Shamburek R.D. Foger B. Chase M. Bensadoun A. Hoyt Jr., R.F. Brewer Jr., H.B. Santamarina-Fojo S. J. Lipid Res. 1998; 39: 2436-2442Abstract Full Text Full Text PDF PubMed Google Scholar, 11Merkel M. Kako Y. Radner H. Cho I.S. Ramasamy R. Brunzell J.D. Goldberg I.J. Breslow J.L. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 13841-13846Crossref PubMed Scopus (137) Google Scholar, 12Dugi K.A. Amar M.J.A. Haudenschild C.C. Shamburek R.D. Bensadoun A. Hoyt R.F.J. Fruchart-Najib J. Madj Z. Brewer H.B.J. Santamarina-Fojo S. Arterioscler. Thromb. Vasc. Biol. 2000; 20: 793-800Crossref PubMed Scopus (59) Google Scholar, 13Zambon A. Deeb S.S. Bensadoun A. Foster K.E. Brunzell J.D. J. Lipid Res. 2000; 41: 2094-2099Abstract Full Text Full Text PDF PubMed Google Scholar). HL and LPL share a number of functional domains such as the Ser-Asp-His catalytic triad, heparin-binding domain, lid region, and lipid- and receptor-binding domains (15Wong H. Davis R.C. Thuren T. Goers J.W. Nikazy J. Waite M. Schotz M.C. J. Biol. Chem. 1994; 269: 10319-10323Abstract Full Text PDF PubMed Google Scholar). Based on their similarity of lipolytic function, amino acid homology, and conservation of disulfide bridges, it is believed that HL and LPL share a similar structure (16Derewenda Z.S. Cambillau C. J. Biol. Chem. 1991; 266: 23112-23119Abstract Full Text PDF PubMed Google Scholar). Despite these similarities, however, differences remain in important enzyme characteristics such as relative heparin affinity, substrate specificity, and cofactor requirements. Unlike HL, LPL requires a specific cofactor, apoC-II, to hydrolyze triglycerides in chylomicrons (17Havel R.J. Shore V.G. Shore B. Bier D.M. Circ. Res. 1970; 27: 595-600Crossref PubMed Scopus (183) Google Scholar, 18LaRosa J.C. Levy R.I. Herbert P. Lux S.E. Fredrickson D.S. Biochem. Biophys. Res. Commun. 1970; 41: 57-62Crossref PubMed Scopus (451) Google Scholar). The importance of apoC-II for LPL function is emphasized by the observation of a significant accumulation of triglycerides in patients who have an inherited defect of the apoC-II gene (19Breckenridge W.C. Little J.A. Steiner G. Chow A. Poapst M. N. Engl. J. Med. 1978; 298: 1265-1273Crossref PubMed Scopus (413) Google Scholar). Initially, the study of chimeric lipases (20Davis R. Wong H. Nikazy J. Wang K. Han Q. Schotz M. J. Biol. Chem. 1992; 267: 21499-21504Abstract Full Text PDF PubMed Google Scholar, 21Dichek H.L. Parrott C. Ronan R. Brunzell J.D. Brewer H.B. Santamarina-Fojo S. J. Lipid Res. 1993; 34: 1393-1401Abstract Full Text PDF PubMed Google Scholar) suggested that a region in the N-terminal domain of LPL was responsible for cofactor activation because enzymes containing the N-terminal domain of LPL and the C-terminal domain of HL were still able to be activated by apoC-II. However, these chimeric enzymes were not activated by apoC-II to the same extent as native LPL. More recently, we reported that the 60 C-terminal amino acids of LPL also participate in apoC-II activation (22Hill J.S. Yang D. Nikazy J. Curtiss L.K. Sparrow J.T. Wong H. J. Biol. Chem. 1998; 273: 30979-30984Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar), suggesting that regions in the N-terminal domain alone are not sufficient to achieve optimal activation. These results are more easily interpreted in the context of a head-to-tail dimer model (15Wong H. Davis R.C. Thuren T. Goers J.W. Nikazy J. Waite M. Schotz M.C. J. Biol. Chem. 1994; 269: 10319-10323Abstract Full Text PDF PubMed Google Scholar, 20Davis R. Wong H. Nikazy J. Wang K. Han Q. Schotz M. J. Biol. Chem. 1992; 267: 21499-21504Abstract Full Text PDF PubMed Google Scholar, 23Wong H. Yang D. Hill J.S. Davis R.C. Nikazy J. Schotz M.C. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 5594-5598Crossref PubMed Scopus (59) Google Scholar, 24Chaillan C. Kerfelec B. Foglizzo E. Chapus C. Biochem. Biophys. Res. Commun. 1992; 184: 206-211Crossref PubMed Scopus (14) Google Scholar, 25Fontana A. Dalzoppo D. Grandi C. Zambonin M. Biochemistry. 1981; 20: 6997-7004Crossref PubMed Scopus (26) Google Scholar), which supports the hypothesis that apoC-II interacts simultaneously with regions located in the N- and C-terminal domains of opposing subunits that make up an LPL dimer (22Hill J.S. Yang D. Nikazy J. Curtiss L.K. Sparrow J.T. Wong H. J. Biol. Chem. 1998; 273: 30979-30984Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). To identify specific LPL amino acid residues that are responsive to cofactor, chemical cross-linking of apoC-II to LPL was undertaken. Cross-linking experiments identified a region from the N-terminal domain of LPL that interacted with apoC-II and whose role in activation was determined using chimeric lipases. The LPL fragment contains two candidate regions, one composed of 4 amino acids and the other of 7, that differ from HL, a highly related but cofactor-unresponsive lipase. A series of chimeras were constructed with the variable regions exchanged between the two lipases, and apoC-II responsiveness was determined. The results suggest that LPL residues 65–68 and 73–79 cooperate in cofactor activation and, moreover, that the functional responsiveness imparted by these LPL residues can be translocated to HL. Overall Strategy—A fragment of human apoC-II spanning residues 44–79 (apoC-II-(44–79)) was chemically cross-linked to purified bovine LPL (Sigma). The mixture was reduced and incubated with o-iodosobenzoic acid, which cleaves proteins at tryptophan and tyrosine residues (24Chaillan C. Kerfelec B. Foglizzo E. Chapus C. Biochem. Biophys. Res. Commun. 1992; 184: 206-211Crossref PubMed Scopus (14) Google Scholar, 25Fontana A. Dalzoppo D. Grandi C. Zambonin M. Biochemistry. 1981; 20: 6997-7004Crossref PubMed Scopus (26) Google Scholar). o-Iodosobenzoic acid-generated peptide fragments were separated on SDS-polyacrylamide gels and identified based on their size and sequence. Step 1—The cross-linker reagent sulfosuccinimidyl-2-(p-azidosalicylamido)ethyl 1,3′-dithiopropionate (SASD) was iodinated using IODO-GEN (Pierce) under conditions recommended by the manufacturer. IODO-GEN (1 mg) was dried in microcentrifuge tubes, and SASD was added (1 mg in 1 ml of 100 mm NaPO4, pH 7.2) together with sodium iodide (100 μCi) and mixed briefly (2 min). The reaction mixture was removed from the tube and desalted to separate unbound radioisotope. Step 2—A cofactor·cross-linker complex composed of apoC-II-(44–79) (100 μm in 100 mm NaPO4, pH 7.2) linked to the iodinated cross-linking reagent SASD was created (Fig. 1). The photolabile azido group in SASD required all steps to be carried out in dimmed room light or within darkened vessels. The cross-linker was used at a 3-fold molar excess over cofactor to maximize linkage via the succinimidyl moiety at neutral pH. Probable sites of apoC-II-(44–79) derivatization included the N terminus and/or basic residues at positions 48, 50, 55, and 76. Excess unbound cross-linker was removed by gel permeation chromatography prior to incubation with LPL. Step 3—The cofactor·cross-linker complex was incubated with bovine LPL (1 mg/ml) in quartz cuvettes at 4 °C. The samples were irradiated for 3 min by an ultraviolet light source placed 4 cm from the cuvettes, with a mirror positioned 2 cm behind. Some experiments contained a "dark control" sample, which was wrapped in foil during irradiation to determine the effect of the absence of UV light exposure. Other samples contained a 50-fold molar excess of unlabeled cofactor to evaluate cross-linking specificity. Step 4 —Following UV radiation, samples were treated with dithiothreitol to a final concentration of 10 mm. Reducing agent was used to sever the disulfide bond in the cross-linker moiety (Fig. 1) and served to transfer the iodine label, originally on the cofactor, to a region of LPL at or near the site of interaction. The cofactor provided the binding specificity and, after covalent attachment, was released by dithiothreitol reduction. The label, now attached to the enzyme, was then traced without further involvement of the cofactor. Typical yields of cross-linking reactions ranged from 2 to 5%. Step 5—Cleavage of LPL was performed with o-iodosobenzoic acid (4 mm). o-Iodosobenzoic acid has been demonstrated to selectively cleave proteins at tryptophan and tyrosine residues under relatively mild conditions (26Mahoney W.C. Hermodson M.A. Biochemistry. 1979; 18: 3810-3814Crossref PubMed Scopus (111) Google Scholar, 27Mahoney W.C. Smith P.K. Hermodson M.A. Biochemistry. 1981; 20: 443-448Crossref PubMed Scopus (73) Google Scholar). Cleavage at tyrosines was eliminated by the inclusion of p-cresol (1 mm), effectively making o-iodosobenzoic acid a tryptophan-specific reagent. SDS-16% polyacrylamide gels were used to separate peptide fragments (nine expected LPL fragments from 8 cleaved tryptophan residues and intact apoC-II (44–79) because it lacks tryptophans). Separated peptides were electrophoretically transferred to polyvinylidene difluoride membranes (Millipore Corp.) for autoradiography and N-terminal sequence analyses. Six chimeras were created that focused on the proposed apoC-II activation site of LPL (residues 65–68 and 73–79) (Fig. 2). Three of the chimeras had the HL backbone with the suspected regions of LPL replacing the corresponding sections of HL. These enzymes were designated HLLPL-(65–68), HLLPL-(73–79), and HLLPLD (where "D" is double chimera). Conversely, the remaining three chimeras consisted of an LPL backbone with the proposed regions exchanged with the corresponding sections of HL. These enzymes were designated LPLHL-(77–80), LPLHL-(85–91), and LPLHLD. To aid in purification of the enzymes, the chimeras and wild-type HL and LPL were constructed with a His6 tag. cDNAs for both wild-type HL and LPL had 6 histidines added to the C-terminal end, and these were used as templates for their respective chimeras. The histidine tag was added to wild-type HL and LPL using primers containing histidine codons. The first PCR consisted of the 5′-histidine primer and a 3′-flanking primer specific for the vector pcDNA3 (5′-HL/His6, CAT CAT CAT CAT CAT CAT TGA GAT TTA ATG AAG ACC CA; 3′-primer/pcDNA3; 5′-LPL/His6, CAT CAT CAT CAT CAT CAT TGA AAC TGG GCG AAT CTA CA; and 3′-primer/pcDNA3). The second PCR contained the 3′-histidine primer and the 5′-flanking primer specific to pcDNA3 (3′-HL/His6, ATG ATG ATG ATG ATG ATG TCT GAT CTT TCG CTT TGA TG; 5′-primer/pcDNA3, AAA TGT CGT AAC AAC TCC GCC; 3′-LPL/His6, ATG ATG ATG ATG ATG ATG GCC TGA CTT CTT ATT CAG AG; and 5′-primer/pcDNA3). The purified products were joined together in a third and final PCR using the flanking primers 5′-primer/pcDNA3 and 3′-primer/pcDNA3 for both HL and LPL. For chimeric construction, restriction endonuclease sites were added to primers defining the 5′ and 3′ termini of the construct to allow for directional cloning. Mutagenic primers (forward and reverse) were designed to span the corresponding boxed coding regions (Fig. 2) and to overlap with one another so that two PCR products could be combined together to form the final full-length cDNA in a third PCR. The primers used for each portion of the chimeras are shown in Table I.Table IPrimers used for chimeric constructionNameSequenceHIND5PKHLACT TAA GCT TGC CAC CAT GGA CAC AAG TCC CCT GTG T1LPLC2HLFORAAC TGG GTG CCA AAA CTT GTG GCC GCG CTG AAG1LPLC2HLREVGGC CAC AAG TTT TGG CAC CCA GTT TTC TAG CACBAM3PHLACG TGG ATC CAA GGA GTA AGA TTC ATT TAT T2LPLC2HLFORTAC AAG AGA GAA CCA GAC TCC GTG ACA GTG GGG CTG2LPLC2HLREVCAC GGA GTC TGG TTC TCT CTT GTA CAG CGC GGC CAC CATD1LPLC2HLFORAAC TGG GTG CCA AAA CTT GTG GCC GCG CTG TACD2LPLC2HLREVCAC GGA GTC TGG TTC TCT CTT GTA CAG CGC GGC CAC AAGHIND5PKLPLACG TAA GCT TGC CAC CAT GGA GAG CAA AGC CCT GCT C1HLC2LPLFORAGT TGG ATC TGG CAG ATG GTG GCC GCC CTG TAC1HLC2LPLREVGGC CAC CAT CTG CCA GAT CCA ACT CTC ATA CATBAM3PLPLACG TGG ATC CGA ATT CAC ATG CCG TTC TTT G2HLC2LPLFORAAG TCT CAG CCG GCC CAG CCA AAT GTC ATT GTG GTG2HLC2LPLREVATT TGG CTG GGC CGG CTG AGA CTT CAG GGC GGC CAC AAGD1HLC2LPLFORAGT TGG ATC TGG CAG ATG GTG GCC GCC CTG AAG Open table in a new tab Full-length cDNAs were purified, digested, and inserted into the pcDNA3 expression vector (Invitrogen) using the HindIII and BamHI restriction endonuclease sites. The DNA sequence was confirmed prior to transfection. Chinese hamster ovary Pro5 cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and antibiotics (Invitrogen). To mediate the transfection of Chinese hamster ovary cells, coprecipitates of plasmid DNA and CaPO4 were prepared (28Chen C. Okayama H. Mol. Cell. Biol. 1987; 7: 2745-2752Crossref PubMed Scopus (4816) Google Scholar). The calcium phosphate/DNA mixture was incubated at room temperature for 30 min before it was added to a 50% confluent Chinese hamster ovary monolayer. Stably transfected cells were selected by growth in the presence of Geneticin (G418 sulfate; 500 μg/ml), and surviving colonies were selected and expanded. Cell clones expressing maximal quantities of lipase were identified by enzyme activity analysis. After growth to confluency in T-175 flasks, the medium was replaced with Opti-MEM (Invitrogen) supplemented with 10 units/ml heparin. The medium was harvested and replaced every 24 h for an 8-day period. After centrifugation at 3000 × g for 10 min to remove cell debris, protease inhibitor mixture for mammalian cell and tissue extracts (Sigma) was added to a final concentration of 0.02 mm, and the harvested medium was stored at –80 °C. All purification steps were carried out at 4 °C. Thawed wild-type or chimeric HL medium (1 liter) was mixed with NaCl to a final concentration of 0.5 m and applied to an octyl-Sepharose column (2.6 × 25 cm) previously equilibrated with 50 mm Tris-HCl, pH 7.2, containing 0.35 m NaCl. Following a wash with 500 ml of 50 mm Tris-HCl, 0.5 m NaCl, 20% glycerol, and 0.02 mm protease inhibitor, pH 7.2 (Buffer A), the lipase was eluted with 500 ml of 50 mm Tris-HCl, 0.35 m NaCl, 20% glycerol, and 0.02 mm protease inhibitor, pH 7.2 containing 1.2% Igepal CA-630 (Sigma) onto a heparin-Sepharose column (2.6 × 25 cm). This column was washed with 500 ml of Buffer A prior to elution with 250 ml of 50 mm Tris-HCl, pH 7.2, 2 m NaCl, 20% glycerol, and protease inhibitor (0.02 mm), onto a 1 × 10-cm metal affinity column (QIAGEN Inc.). The column was washed with 25 ml of Buffer A before elution with 26 ml of 50 mm Tris-HCl, 0.5 m NaCl, and 250 mm imidazole, pH 7.2. The eluent was collected in eight fractions, the first one being 5 ml and the rest 3 ml. Each fraction was assayed for activity, and the active fractions were concentrated in a Millipore filtration unit (molecular mass cutoff of 100,000 Da) to a final volume of ∼1 ml and stored at –80 °C. Wild-type LPL and the LPL chimeras were purified in the same manner with two exceptions. 1) The octyl-Sepharose step was omitted; therefore, the thawed medium was loaded directed onto the heparin-Sepharose column with no NaCl added. 2) Buffer A contained NaCl at 0.75 m, not 0.5 m. The purity of the enzyme preparation was determined by densitometry of silver-stained SDS-polyacrylamide gels. Trioleinase activity was measured using a triolein emulsion containing radiolabeled triolein as described previously (22Hill J.S. Yang D. Nikazy J. Curtiss L.K. Sparrow J.T. Wong H. J. Biol. Chem. 1998; 273: 30979-30984Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). ApoC-II-dependent lipase activity was determined by performing the assay in the presence of an apoC-II fragment spanning residues 44–79. This apoC-II fragment has been shown to have the same activating potential as full-length apoC-II (29Kinnunen P.K.J. Jackson R.L. Smith L.C. Gotto Jr., A.M. Sparrow J.T. Proc. Natl. Acad. Sci. U. S. A. 1977; 74: 4848-4851Crossref PubMed Scopus (128) Google Scholar). Protein concentration was measured by a colorimetric assay developed by Smith et al. (30Smith P. Krohn R. Hermanson G. Mallia A. Gartner F. Provenzano M. Fujimoto E. Goeke N. Olson B. Klenk D. Anal. Biochem. 1985; 150: 76-85Crossref PubMed Scopus (18512) Google Scholar) using a Pierce micro-BCA protein assay reagent kit. Kinetic constants were determined using GraphPAD Prism Version 3.02 for Windows. Gels were fixed in 100 ml of 30% ethanol and 10% glacial acetic acid for 30 min and then washed twice with 10% ethanol and three times with deionized water for 5 min/wash. The gels were soaked in 50 ml of SilverSNAP stain solution with 1 ml of SilverSNAP enhancer solution (Pierce) for 30 min with gentle shaking. The developer was removed, and the gels were washed with deionized water for 30 s. The gels were transferred to 50 ml of SilverSNAP developer with 1 ml of SilverSNAP enhancer for developing until bands appeared. Samples were mixed with 0.5 volume of buffer containing 2% SDS, 0.1 m Tris-HCl, pH 6.8, 50% glycerol, 10% β-mercaptoethanol, and 0.05% bromphenol blue. The mixture was placed in boiling water for 5 min prior to loading onto a 10% acrylamide gel. Gels were electroblotted onto a polyvinylidene difluoride hydrophobic membrane that was pretreated with 100% methanol for 10 s. The membrane was placed on filter paper and air-dried for 15 min. The blot was placed in 15 ml of 1% casein and 0.04% Tween 20 (antibody buffer) containing either a monoclonal antibody specific for human HL (22Hill J.S. Yang D. Nikazy J. Curtiss L.K. Sparrow J.T. Wong H. J. Biol. Chem. 1998; 273: 30979-30984Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar) or a chicken polyclonal antibody raised against bovine LPL (a kind gift from Dr. O. Ben-Zeev) and incubated for 1 h. The blot was rinsed with PBS and washed for 5 min with fresh PBS, which was then repeated twice. Immunoblotting with the monoclonal or polyclonal antibody was detected with either anti-mouse IgG or anti-chicken IgG conjugated to biotin in 15 ml of antibody buffer for 20 min. After washing, the blot was incubated with streptavidin conjugated to horseradish peroxidase in PBS with 0.1% Triton X-100 for 10 min. The blot was developed with chemiluminescent reagents (Pierce) and exposed to chemiluminescent film (Amersham Biosciences). 200 μl of anti-LPL antibody 5D2 (a kind gift from Dr. John D. Brunzell) (31Babirak S.P. Iverius P.H. Fujimoto W.Y. Brunzell J.D. Arterioscler. Thromb. Vasc. Biol. 1989; 9: 326-334Google Scholar) was added to each well of a Costar high binding enzyme immunoassay/radioimmunoassay plate at a dilution of 4 μg/ml as previously described (32Peterson J. Fujimoto W.Y. Brunzell J.D. J. Lipid Res. 1992; 33: 1165-1170Abstract Full Text PDF PubMed Google Scholar). The plate was sealed with a Mylar plate sealer and incubated at 37 °C for 4 h. The plate was washed three times with PBS and 0.05% Tween 20. After the third wash, 300 μl of PBS/Tween 20 was added per well, and the plate was then sealed and left overnight at 4 °C. The buffer was removed from the plate, and standards (purified bovine LPL) diluted to 0.1 μg/μl in 50% glycerol and 10 mm NaH2PO4 at pH 7.5, then 4 μl of this solution diluted in 796 μl of 4.56% bovine serum albumin in PBS to 0.5 ng/μl, controls (heparin challenge plasma), and samples (LPL, LPLHL-(77–80), LPLHL-(85–91), and LPLHLD) were added to the plate in quadruplicates at 200 μl/well. The plate was sealed and incubated overnight at 4 °C. Plates were washed four times with PBS/Tween 20; 200 μ of 5D2 peroxidase solution (100 ml of PBS, 100 μl of Tween 20, and 50 μl of 5D2 peroxidase) was added per well; and the plate was sealed and incubated at room temperature for 4 h. The plate was washed five times with PBS/Tween 20. In a darkened room, 200 μl of substrate (75 ml of citrate buffer, pH 5.0, 30 mg of o-phenylenediamine tablet (Sigma), and 150 μl of 3% hydrogen peroxide) was added per well, and the plate was covered and incubated in the dark at room temperature for 10–20 min (the absorbance of the highest standard was 0.600–0.800). 50 μl of 4 m H2SO4 was added per well to stop color development, and the absorbance at 492 nm was determined. The model of human LPL was generated using, as a template, the 2.46-Å resolution structure of the human pancreatic lipase·colipase complex inhibited by a C11 alkyl phosphonate (Protein Data Bank code 1LPB) (33Egloff M.P. Sarda L. Verger R. Cambillau C. van Tilbeurgh H. Protein Sci. 1995; 4: 44-57Crossref PubMed Scopus (57) Google Scholar), which has 30% homology to human LPL. The model was created using the 3D-JIGSAW algorithm (34Bates P.A. Sternberg M.J. Proteins. 1999; 3: 47-54Crossref PubMed Scopus (148) Google Scholar) (amino acids 1–434 of the mature LPL sequence were modeled) and viewed/analyzed using Swiss-PdbViewer (35Guex N. Peitsch M.C. Electrophoresis. 1997; 18: 2714-2723Crossref PubMed Scopus (9507) Google Scholar). Binding of ApoC-II to LPL—Binding of the cofactor·cross-linker complex to LPL was assessed by its ability to stimulate lipase activity. The complex stimulated LPL activity to a similar extent as serum (∼7-fold) (data not shown), indicating that the presence of the cross-linker had little effect on lipase function. Furthermore, four of the five possible sites of linkage between apoC-II and the cross-linker (residues 44, 48, 50, and 55) are not found in the region of apoC-II believed to activate LPL (36Storjohann R. Rozek A. Sparrow J.T. Cushley R.J. Biochim. Biophys. Acta. 2000; 1486: 253-264Crossref PubMed Scopus (18) Google Scholar). Thus, it was concluded that the cofactor·cross-linker complex was suitable to probe the cofactor-binding site of LPL. LPL was incubated with the cofactor·cross-linker complex and then photolyzed, reduced, cleaved, and displayed on denaturing gels (Fig. 3). This autoradiograph of an SDS-16% acrylamide gel shows a single radiolabeled peptide fragment, which migrated below the 3.5-kDa standard, but above the dye front. Lane 2 shows the pattern of another sample run under identical conditions, except for the inclusion of a 50-fold excess of unlabeled apoC-II, and the reduction step was omitted. In this case (as for dark control samples), a 3.5-kDa band was seen; no band was detected corresponding to the band in lane 1. The band migrating at 3.5 kDa corresponded to the size of the apoC-II·cross-linker complex, whose identity was confirmed by microsequence analyses (data not shown). Thus, the 3.5-kDa band in lane 2 is the unbound cofactor·cross-linker complex; and significantly, inclusion of excess unlabeled apoC-II completely eliminated the lower band (Fig. 3, compare lanes 1 and 2), suggesting specific interaction between apoC-II and this portion of LPL. The mass of the labeled peptide (Fig. 3, lane 1) was determined to be 2.2 kDa by comparison with the migration positions of proteins with known molecular masses. Based on the locations of the 8 tryptophan residues in bovine LPL, the molecular mass of the labeled LPL peptide most closely corresponds to that of peptide 3, from residues 65 to 86 (Table II). This conclusion was supported by the determination of a valine residue at the N terminus of the labeled peptide (data not shown); only the sequence of peptide 3 begins with a valine residue.Table IISizes of bovine LPL fragments following cleavage at tryptophan residuesFragment no.Amino acid residuesMolecular mass DaN-terminal domain peptide11-556050256-64990365-862420487-1143080N- and C-terminal domain peptide5115-38229,500C-terminal domain peptide6383-3908807391-3933308394-3951109396-4485940 Open table in a new tab Construction of ApoC-II Activation Site Chimeras—Upon comparison of residues 65–86 of LPL with the corresponding region in HL, two regions of dissimilarity were identified (Fig. 2). To determine whether these sequences are associated with the abi
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