Studies on the immunological cross-reactivity of various pancreatic colipases. Isolation by immunoaffinity chromatography of a single form of procolipase from porcine pancreas

Antibodies against porcine procolipase B were produced in rabbits. The antiserum was used to immunoinactivate various forms of native and trypsin-treated porcine colipase. Our results indicate that all forms of the porcine cofactor bind to anti-porcine procolipase B antibodies. Human colipase showed lower affinity for the antibodies than porcine colipase. No cross-reactivity was observed between pig and horse, cow, dog or chicken colipases. Immunological studies on porcine colipase, carried out in the presence of lipid, provided evidence that antibodies bind to colipase at or near the lipase binding site. The binding of antibodies to colipase is not affected by the adsorption of the cofactor at a lipid interface. Using a predictive method for identification of the antigenic determinants, it was found that, in pig colipase, regions at positions 42-48 and 70-74 might represent antigenic sites. In the horse protein, the peptide segment 42-48 was also recognized as a possible antigenic site. An immunoadsorbent gel column was prepared for a one-step isolation of porcine colipase. In contrast to purification methods described so far, immunoaffinity chromatography yielded only one form of the porcine cofactor when starting from a pancreatic extract. This protein preparation has structural, biochemical and immunochemical properties similar to that of porcine procolipase A previously isolated from pancreas in the presence of detergent.     

Inhibitory properties and antigenic specificity of monoclonal antibodies to pancreatic colipase

To understand the mechanism by which colipase acts as a protein cofactor for anchoring pancreatic lipase at triacylglycerol/water interface, we have used an immunochemical approach. Ten monoclonal antibodies (Mabs) against porcine pancreatic procolipase were produced. Purified immunoglobulins and Fab fragments were studied for their capacity to inhibit colipase-dependent lipase activity. These studies were carried out by using procolipase, the secretory form of the cofactor, and its trypsin-treated form obtained by removal of the amino terminal pentapeptide by trypsin. Reactivities of Mabs with both forms of the cofactor were also studied by immunoenzymatic methods. Mabs 6.1, 49.20. 75.8, 270.13 and 419.1 were found to inhibit lipolysis by preventing the binding of procolipase or trypsin-treated colipase to the lipid substrate. Mab 72.11 inhibited procolipase binding but had no effect on trypsin-treated colipase. Mab 72.11 reacted with procolipase in ELISA but showed no reactivity with trypsin-treated colipase. Finally, preincubation of Mab 72.11 with porcine procolipase prevented specific cleavage at the Arg5-Gly6 bond by trypsin. It could be concluded, that the five first residues of procolipase are structural elements of the antigenic determinant recognized by Mab 72.11. Results of ELISA additivity tests (cotitrations) further indicated that epitopes for Mabs 6.1, 72.11, 270.13 and 419.1 and for Mabs 49.20 and 75.8 are located in two distinct antigenic regions of the procolipase molecule. It appears then that the lipid binding domain of the pancreatic lipase protein cofactor comprises two regions. The first region corresponds to the amino terminal fragment of the protein. The second region is likely identical with the peptide segment at position 51-59 as previously hypothesized from NMR and spectrophotometric studies. Studies carried out on procolipase chemically modified at tyrosine residues provided evidence that epitopes for Mabs 49.20 and 75.8 are in or close to the region which contains tyrosines at positions 55 and 59, and that the two peptide regions essential for interfacial binding are spatially adjacent in the procolipase and the trypsin-treated form of the cofactor. General conclusions are in accordance with the location of antigenic regions of procolipase determined by predictive methods.     

Inhibition of pancreatic colipase by antibodies and Fab fragments. Selective effects of two fractions of antibodies on the functional sites of the cofactor

Rabbit antiserum was raised against porcine pancreatic colipase and Fab fragments were prepared by papain digestion of purified antibodies followed by purification on protein A-Sepharose. Fab fragments showed inactivation toward porcine colipase activity similar to that of antiserum and purified antibodies. From inactivation studies carried out by incubating porcine colipase and lipase with Fab fragments in the absence of lipid or in the presence of triolein and sodium deoxycholate, it could be concluded that polyclonal antiporcine colipase antibodies contain fractions that bind specifically to epitopes at or near the functional regions of the porcine cofactor. Studies with an enzyme-linked immunosorbent assay showed that cross-reactivity of horse or chicken colipase with antiporcine colipase antiserum was lower than that of the human or porcine protein. Results of immunoactivation kinetic studies performed with the same proteins, fully confirmed these observations. Partial cross-reactivity between porcine and chicken colipases allowed us to fractionate antibodies by immunoaffinity chromatography on immobilized chicken colipase. Fraction I contains antibodies absorbed on porcine colipase not accessible when the cofactor is bound to lipid. Antibodies of fraction II, nonadsorbed on chicken colipase, inactivate porcine colipase preincubated with triolein/deoxycholate. Lipase had a protective effect against inactivation. Antibodies of fraction II bind likely to epitopes close to the specific region of colipase interacting with lipase. Our conclusions are in good agreement with analysis of the sequence of porcine, equine and human colipases by calculating local hydrophilicity indices.     

Production and characterization of four monoclonal antibodies against porcine pancreatic colipase

Four monoclonal antibodies directed against porcine colipase have been generated by hybridization of myeloma cells with spleen cells of BALB/c immunized mice. Antibodies were screened by binding to immobilized colipase in a solid-phase assay. Monoclonal antibodies were purified by affinity chromatography on colipase coupled to Sepharose. All monoclonal antibodies are of the IgG1 class with high affinity for the antigen. The dissociation constant of the complex formed in solution between porcine colipase and antibody varied from 1.1 X 10(-10) M to 1.8 X 10(-8) M. Epitope specificity was studied for each antibody and in pairs with an enzyme-linked immunosorbent assay (ELISA). Results indicate that the four monoclonal antibodies react with at least three different antigenic regions of colipase. Finally, three monoclonal antibodies were found to be potent inhibitors of colipase activity. Antiporcine monoclonal antibodies appear to be suitable probes for studying the lipid affinity site of the protein cofactor of pancreatic lipase.     

The histidine residues in pig and horse colipases

The single histidine of horse colipase B was shown to be at position 29, thus definitely proving that His₈₆ present in the pig cofactor is not conserved in the horse. Carbethoxylation of histidines did not appreciably affect the capacity of the porcine cofactor to bind to hydrophobic interfaces and to anchor lipase in presence of bile salts, but it induced in the aromatic region of the molecule a significant transconformation detectable by spectrofluorimetry.     

Antigen specificity and cross-species reactivity of a monoclonal antibody (mAb 72.11) against porcine pancreatic procolipase.

We have studied the antigen specificity and cross-reactivity of a monoclonal antibody (mAb 72.11) of subclass IgG1, raised against the precursor form of porcine colipase (procolipase), whose epitope lies near the amino terminal region of the polypeptide. mAb 72.11 cross-reacts with native porcine, equine and human procolipase, as shown by immuno-inactivation and ELISA titration studies carried out on pure proteins, pancreatic tissue homogenate or pancreatic juice. The epitope site recognized by mAb 72.11 was further characterized by studying antibody binding to denatured procolipase. Reduced carboxymethylated procolipase reacted with mAb 72.11 in ELISA. Heat inactivated or reduced carboxymethylated porcine procolipase displaced antigen from the complex formed between antibody and native procolipase. The lack of sensitivity of epitope recognized by mAb 72.11 on procolipase to heat denaturation or reduction of the disulfide bridges is indicative that antigen specificity of mAb 72.11 is not dependent on the conformation of the antigenic site. Cross-reactivity of mAb 72.11 with procolipase from the three species demonstrates that substitution of amino acid at positions 1 and 3 causes no loss of antigenicity. Finally, mAb 72.11 was coupled to sepharose to isolate human procolipase from human pancreatic juice and to separate the precursor form from activated colipase non-adsorbed on the column.     

The primary sequence of human pancreatic colipase

The amino acid sequence of an activated colipase purified from human pancreas was determined. The protein consists of a single polypeptide chain of 86 amino acids (human colipase₈₆) and has a molecular weight of 9289. The sequence was determined by automated Edman degradation of the reduced and S-carboxymethylated protein and of two CNBr peptides. Sequence determination of porcine procolipase II was also performed, which showed that in the original sequence determination apparently two residues were missed. These residues were determined to be a leucine at position 37 and a serine in position 50. For comparison with porcine and equine procolipases, the residues composing human colipase are numbered from 6 to 91. No human procolipase has been isolated so far. The colipases from man, pig, horse and chicken show a high degree of homology: human colipase differs from the other proteins by substitutions of 19 (porcine), 24 (equine A) and 21 (equine B) residues, respectively.     

Evidence for the existence of procolipase in chicken pancreas and pancreatic juice

Purified antibodies raised against chicken colipase were coupled to Sepharose 4B and colipase was isolated in a single step by immunoaffinity chromatography from an extract of chicken pancreas prepared under conditions where trypsin activation is avoided. The purified protein has a single amino terminal residue of alanine and its biochemical properties are similar to those of the precursor form of colipase (procolipase) previously isolated from porcine and equine pancreas or pancreatic juice. Further evidence for the existence of procolipase was obtained from kinetic studies of the hydrolysis of the Intralipid emulsion by untreated and trypsin-treated chicken pancreatic juice.     

Uncoupling of catalysis and colipase binding in pancreatic lipase by limited proteolysis.

In the intestine, the hydrolysis of triglycerides by pancreatic lipase is performed only in the presence of colipase, whose function is to anchor lipase to the bile-salt-coated lipid interface. Biochemical and crystallographic data on porcine and human lipases have shown that the molecule is made of two well-delimited domains. In order to get more information on the role of the domains in catalysis and colipase binding, we performed limited proteolysis on lipase from various species and obtained different patterns of cleavage. In the case of porcine and human lipases, only the C-terminal domain (12 kDa) could be obtained after chymotryptic attack, whereas in the horse enzyme the cleavage of the Leu410-Thr411 bond gave rise to a large N-terminal (45 kDa) and a small C-terminal (4 kDa) fragment. The isolated porcine and human C-terminal domains were completely inactive towards emulsified tributyrin, though were able to bind colipase. Conversely, the horse 45 kDa fragment retained the lipase activity but failed to correctly bind colipase. This work definitely proves that catalysis and colipase binding are separate events involving topographically distinct regions of the molecule and focuses attention on the role of the C-terminal domain in colipase binding.     

Isolation and characterization of colipase from porcine and human pancreatic juice by immunoaffinity chromatography

Pure colipase was prepared by immunoaffinity chromatography from porcine and human pancreatic juice. A single form of the porcine colipase was obtained, having the structural and biological properties of previously characterized porcine procolipase A. Two forms of activated colipase (N-terminal Gly) were isolated from human pancreatic juice by the same procedure. The existence of two forms of activated colipase might arise from rapid activation of a precursor form of human colipase during collection of the pancreatic juice.     

The effect of pancreatic procolipase and colipase on pancreatic lipase activation

Intestinal fat digestion is carried out by the concerted action of pancreatic lipase and its protein cofactor colipase. Colipase is secreted from pancreas as a procolipase and is transformed into colipase by the trypsin cleavage of the Arg5-Gly6 bond during liberation of an N-terminal pentapeptide. The kinetic parameters for the lipase-colipase system compared to the lipase-procolipase system has been compared using trioctanoin and Intralipid as substrates. It was found that at pH 7.0 the Kmapp using Intralipid as substrate was the same for procolipase and colipase, 0.06 mM and 0.05 mM, respectively. At pH 8.0, however, the Kmapp were different-0.23 mM for procolipase and 0.08 mM for colipase. In a similar way the binding between colipase and lipase had a dissociation constant of 2.4 x 10(-6) M at pH 7.0, while for procolipase--lipase binding the dissociation constant was 4.1 x 10(-6) M with no significant difference. At pH 8.0 the binding between colipase and lipase was stronger, Kd being 2.0 x 10(-7) M, while weaker for procolipase and lipase, Kd being 1.0 x 10(-5) M. It is concluded that at the physiological pH value as is found in the intestine, the activation of procolipase to colipase has no influence on the hydrolysis of trioctanoin or Intralipid in the presence of bile salt.     

Separation and characterization of the precursor and activated forms of porcine and human pancreatic colipase by reversed-phase liquid chromatography

Reversed-phase liquid chromatography was used as an alternative method for the characterization of the precursor and activated forms of porcine and human pancreatic colipase. Using a Beckman Ultrasphere column with an increasing acetonitrile gradient in 0.1% trifluoroacetic acid, it was possible to obtain well-resolved separation of the precursor form of colipase (procolipase) from its trypsin-activated derivative. This protocol was used (1) to study the activation of porcine procolipase by trypsin or thrombin in vitro, (2) to assess the homogeneity of porcine colipase preparations used in tridimensional structure studies and in combination with immunoaffinity chromatography, (3) to identify the form of colipase present in samples of human pancreatic juice.     

Characterization of triton X 100 extracted colipase from porcine pancreas

Colipase was isolated from porcine pancreas homogenate prepared in the presence of detergent (Triton X 100). After precipitation by ammonium sulfate and ethanol, the cofactor was purified by chromatography on SP-Sephadex in the presence of Triton X 100 and on DEAE-cellulose in the absence of detergent. Two molecular forms of porcine colipase were obtained. They represent 80 per cent (colipase A) and 20 per cent (colipase B), respectively, of the total colipase. Valine is the N-terminal residue of both proteins. Their aminoacid composition is similar to that found by Borgstrom for the two forms of porcine colipase. Determination of the sequence of the first sixteen residues at the N-terminal end of colipase A indicates that the cofactor undergoes no proteolytic degradation in this region of the molecule when extraction is carried out in the presence of detergent. The recovery of colipase is about 30 per cent.     

Characterization of Triton X 100 extracted colipase from porcine pancreas

Colipase was isolated from porcine pancreas homogenate prepared in the presence of detergent (Triton X 100). After precipitation by ammonium sulfate and ethanol, the cofactor was purified by chromatography on SP-Sephadex in the presence of Triton X 100 and on DEAE-cellulose in the absence of detergent. Two molecular forms of porcine colipase were obtained. They represent 80 per cent (colipase A) and 20 per cent (colipase B), respectively, of the total colipase. Valine is the N-terminal residue of both proteins. Their aminoacid composition is similar to that found by Borgstrom for the two forms of porcine colipase. Determination of the sequence of the first sixteen residues at the N-terminal end of colipase A indicates that the cofactor undergoes no proteolytic degradation in this region of the molecule when extraction is carried out in the presence of detergent. The recovery of colipase is about 30 per cent.     

The affinities of procolipase and colipase for interfaces are regulated by lipids.

It has been suggested that at physiological pH, the trypsin-catalyzed activation of the lipase cofactor, procolipase, to colipase has no consequence for intestinal lipolysis and serves primarily to release the N-terminal pentapeptide, enterostatin, a satiety factor (Larsson, A., and C. Erlanson-Albertsson 1991. The effect of pancreatic procolipase and colipase on pancreatic lipase activation. Biochim. Biophys. Acta 1083:283-288). This hypothesis was tested by measuring the adsorption of [14C]colipase to monolayers of 1-stearoyl-2-oleoyl-sn-3-glycerophosphocholine and 13, 16-cis, cis-docosadienoic acid in the presence and absence of procolipase. With saturating [14C]colipase in the subphase, the surface excess of [14C]colipase is 29% higher than that of procolipase, indicating that colipase packs more tightly in the interface. With [14C]colipase-procolipase mixtures, the proteins compete equally for occupancy of the argon-buffer interface. However, if a monolayer of either or both lipids is present, [14C]colipase dominates the adsorption process, even if bile salt is present in the subphase. If [14C]colipase and procolipase are premixed for > 12 h at pH approximately 8, this dominance is partial. If they are not premixed, procolipase is essentially excluded from the interface, even if procolipase is added before [14C]colipase. These results suggest that the tryptic cleavage of the N-terminal pentapeptide of procolipase may be of physiological consequence in the intestine.     

A spectrophotometric assay for the characterization of the S'' subsite specificity of alpha-chymotrypsin

Pig and horse colipases contain three tyrosine residues. In addition, horse colipase possesses a tryptophan residue. Some of the tyrosine residues are involved in the association of colipase and a bile salt micelle. The present report demonstrates that the aromatic residues responsible for colipase fluorescence are in an aqueous environment. In the presence of bile salt micelles, changes in colipase fluorescence properties indicate that the intrinsic fluorophores are located in a more hydrophobic environment upon colipase-micelle complex formation. In addition, the fluorescence of an NBD group fixed on lysine 60, which is very close to the aromatic region in the pig colipase, is also altered in the presence of micelles. These results show that the micelle binding site is not limited to the tyrosine residues but may be broadened to adjacent residues such as lysine 60 and also tryptophan 52 in horse colipase.     

Isolation and partial characterization of bovine pancreatic colipase.

Three molecular forms of colipase (colipases A, B and C) with the same specific activity have been isolated from an acid extract of bovine pancreas. Purification includes ammonium sulfate precipitation, ethanol treatment, chromatography on SP-Sephadex, chromatography on DEAE-cellulose and chromatography on QAE-Sephadex. The most basic form of bovine colipase (colipase A) has a molecular weight of 11,000-12,000 daltons and contains 104 residues. Its aminoacid composition is very similar to that of the intact form of porcine colipase isolated by Borgstr?m et al. Colipases from both species have the same N-terminal residue (valine). It is likely that bovine colipases B and C represent partially degraded forms of colipase A. Their cofactor activity, however, is the same.     

Cloning and characterization of the human colipase cDNA

Pancreatic lipase hydrolyzes dietary triglycerides to monoglycerides and fatty acids. In the presence of bile salts, the activity of pancreatic lipase is markedly decreased. The activity can be restored by the addition of colipase, a low molecular weight protein secreted by the pancreas. The action of pancreatic lipase in the gut lumen is dependent upon its interaction with colipase. As a first step in elucidating the molecular events governing the interaction of lipase and colipase with each other and with fatty acids, a cDNA encoding human colipase was isolated from a lambda gt11 cDNA library with a rabbit polyclonal anti-human colipase antibody. The full-length 525 bp cDNA contained an open reading frame encoding 112 amino acids, including a 17 amino acid signal peptide. The predicted protein sequence contains 100% of the published protein sequence for human colipase determined by chemical methods, but predicts the presence of five additional NH2-terminal amino acids and four additional COOH-terminal amino acids. Comparison of the predicted protein sequence with the known sequences of colipase from other species reveals regions of extensive identity. In vitro translation of mRNA transcribed from the cDNA gave a protein of the expected molecular size that was processed by pancreatic microsomal membranes. Sequence analysis of the in vitro translation product after processing demonstrated signal peptide cleavage and the presence of a human procolipase, as exists in the pig and horse colipases. DNA blot analysis was consistent with the presence of a single gene for colipase. RNA blot analysis demonstrated tissue-specific expression of colipase mRNA in the pancreas. Thus, we report, for the first time, a cDNA for colipase.(ABSTRACT TRUNCATED AT 250 WORDS)     

The activation peptide of pancreatic procolipase decreases food intake in rats

Pancreatic procolipase is a cofactor for lipase and necessary for optimal fat digestion in the intestine during a meal. It is activated by trypsin in the intestine during release of an activation peptide, with the sequence Val-Pro-Asp-Pro-Arg in rat. This peptide, in the following termed VPDPR, was found to decrease food intake in rats. The human procolipase activation peptide with the sequence Ala-Pro-Gly-Pro-Arg (APGPR) had no effect on food intake in rats, nor the trypsinogen activation peptide with the sequence Phe-Pro-Val-Asp-Asp-Asp-Asp-Lys (FPVDDDDK). Procolipase added to standard pellets decreased the daily food intake in rats, whereas colipase added to pellets had no effect.     

Computational study of colipase interaction with lipid droplets and bile salt micelles

Colipase is a key element in the lipase-catalyzed hydrolysis of dietary lipids. Although devoid of enzymatic activity, colipase promotes the pancreatic lipase activity in physiological intestinal conditions by anchoring the enzyme at the surface of lipid droplets. Analysis of structures of NMR colipase models and simulations of their interactions with various lipid aggregates, lipid droplet, and bile salt micelle, were carried out to determine and to map the lipid binding sites on colipase. We show that the micelle and the oil droplet bind to the same side of colipase 3D structure, mainly the hydrophobic fingers. Moreover, it appears that, although colipase has a single direction of interaction with a lipid interface, it does not bind in a specific way but rather oscillates between different positions. Indeed, different NMR models of colipase insert different fragments of sequence in the interface, either simultaneously or independently. This supports the idea that colipase finger plasticity may be crucial to adapt the lipase activity to different lipid aggregates.