Purification and characterization of human pancreatic colipase.

Two colipases, named colipase I and colipase II, have been isolated from extracts of human pancreatic gland. The two proteins can be separated by ion-exchange chromatography, isoelectric focusing and slab technique gel electrophoresis. The result of this study indicates that the two colipases, both of which are glycoproteins, have identical amino acid compositions. The pI values were found to be 6.1 for colipase I and 5.8 for colipase II. The different colipases have also been found in human pancreatic juice. The N-terminal amino acid was glycine for both colipase I (gland) and colipase II (juice). Only minor differences were found between the colipases isolated from gland and juice, and colipase I from gland alone was examined in detail.     

Evolutionary studies on pancreatic colipase

In this evolutionary study the following criteria have been used to prove the existence of colipase: 1. It restores the activity of human and porcine pancreatic lipase inhibited by bile salt. 2. It cross-reacts with antisera to human and porcine colipases. 3. Its restoration of lipase activity, inhibited by bile salt, in the tributyrin assay system, is prevented by antiserum to colipase. 4. It is a heat-stable, low-molecular-weight protein (molecular weight about 10 000 by gel-filtration). The occurrence of colipase has been verified in the exocrine pancreatic cells from hagfish (Myxine glutinosa), ratfish (Chimaera monstrosa), rayfish (Raja radiata), Greenland shark (Somnius microcephalus) and dogfish (Squalus acanthius). No colipase activity could be found in the gastric juice of crayfish (Pacifastacus leniusculus). These results indicate that colipase envolved in the vertebrates before the organized exocrine pancreatic gland and occurred simultaneously with the bile salts/bile alcohols.     

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 two isocolipases in horse pancreas

The N-terminal amino acid sequences of two forms of colipase isolated from horse pancreas have been compared. Four sequence differences were found in the first 51 amino acids. This lead us to conclude that there are two distinct colipases in the horse pancreas.     

Rat pancreatic colipase mRNA: nucleotide sequence of a cDNA clone and nutritional regulation by a lipidic diet

A cDNA clone encoding rat pancreatic colipase was isolated using as a probe a synthetic deoxyoligonucleotide corresponding to a highly conserved amino acid sequence region in colipases from other species. The cloned messenger codes for a protein of 95 amino acids plus a signal peptide of 17 amino acids. The structure of the full-length cDNA was also determined and the corresponding amino acid sequence showed a high degree of homology with those of other known colipases. Quantification of the homologous mRNA in the pancreas of animals fed a high-lipid diet was consistent with a specific though moderate induction of colipase messenger by the nutritional manipulation.     

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)     

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.     

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.     

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.     

Conformational prediction studies on pancreatic colipase.

Comparison of the primary structures of pancreatic colipases from man, pig, horse and rat shows a high degree of homology between proteins. Fifty-two out of the 95 residues of the polypeptide are identical. All colipases contain 10 half-cystines which are located at invariant positions. The secondary structure of colipases has been predicted from the sequence using the statistical method of Chou and Fasman and the method of Gibrat, Garnier and Robson based on information theory. Predictions indicate that colipases have a low content of alpha-helix and beta-strand structure. The two segments at positions 7-10 and 56-59, assumed to be part of the lipid binding domain, have predicted beta-sheet conformation and should be in close spatial vicinity to each other in the proteins. Four beta-turns are predicted in all colipases at positions 3-6, 46-49, 61-64, and 81-84. They might contribute, with the five disulfide bridges, to a tight packing of the protein molecule. Surface residues and major sequential antigenic determinants of mammalian colipases have been predicted using methods based either on hydrophilicity/hydropathy scales or amino acid mutability. From these studies, it appears that colipases exhibit large conformational homologies. In the absence of data on the tertiary structure of colipase, predictive methods, together with physico-chemical and immunological studies, provide valuable information on the conformation of the protein in relation to the topology of residues involved in the functional and antigenic sites.     

Biochemical and structural comparative study between bird and mammal pancreatic colipases

Three colipases were purified from pancreas of two birds (ostrich and turkey) and one mammal (dromedary). After acidic and/or heat treatment and precipitation by sulfate ammonium and then ethanol, cofactors were purified by Sephadex G-50 gel filtration followed by ion-exchange chromatography first on Mono S and then on Mono Q. One molecular form was obtained from each species with a molecular mass of approximately 10 kDa. Cofactors were not glycosylated. The N-terminal sequences of the three purified cofactors showed high sequence homology. A 90 amino acid sequence of the ostrich cofactor was established based on peptide sequences from four different digests of the denaturated protein using trypsin, chymotrypsin, thermolysin, or staphylococcal protease. This sequence exhibited a high degree of homology with chicken and mammal cofactors. Bile salt-inhibited pancreatic lipases from five species were activated to variable extents by colipases from bird and mammal origins. The bird pancreatic lipase-colipase system appears to be functionally similar to homologous lipolytic systems from higher mammals. Our comparative study showed that mammal colipase presents a lower activation level toward bird lipases than the bird counterpart. Three-dimensional modeling of ostrich colipase suggested a structural explanation of this fact.     

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.     

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.     

Fatty acids generated by gastric lipase promote human milk triacylglycerol digestion by pancreatic colipase-dependent lipase

The concerted action of purified bovine gastric lipase and human pancreatic colipase-dependent lipase and colipase, or crude human pancreatic juice, in the digestion of human milk triacylglycerols was explored in vitro. Gastric lipase hydrolyzed milk triacylglycerol with an initially high rate but became severely inhibited already at low concentration of released fatty acid. In contrast, colipase-dependent lipase could not, by itself, hydrolyze milk triacylglycerol. However, a short preincubation of milk with gastric lipase, resulting in a limited lipolysis, made the milk fat triacylglycerol available for an immediate and rapid hydrolysis by pancreatic juice, and also for purified colipase-dependent lipase, provided colipase and bile salts were present. The same effect was obtained when incubation with gastric lipase was replaced by addition of long-chain fatty acid. Long-chain fatty acid increased the binding of colipase-dependent lipase to the milk fat globule. Binding was efficient only in the presence of both fatty acid and colipase. We conclude that a limited gastric lipolysis of human milk triacylglycerol, resulting in a release of a low concentration of long-chain fatty acids, is of major importance for the subsequent hydrolysis by colipase-dependent lipase in the duodenum.     

Comparative studies of canine colipase and lipases from bovine, porcine, canine, human and rat pancreases.

1. Colipase was purified from canine pancreatic juice and found to have certain specificity in its reaction with various pancreatic lipases. 2. This colipase will stimulate the lipolytic activities of lipases isolated from canine, bovine and porcine pancreas but not lipases from a fungus, or from human and rat pancreases. 3. Characterization of these lipases showed (a) the molecular dimension of rat lipase is very different from the other lipases; (b) the pIs of canine, porcine and bovine lipases are almost identical but different from the pIs of rat, human and Candida (a fungus) lipases; and (c) the antiserum prepared against canine lipase will also react with lipases from human, hog and cow pancreases but not with rat and Candida lipases. 4. These physical differences can explain partly the difference in reaction between the various lipases and the canine colipase.     

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.     

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.     

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 immunoads orbent 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.     

Identification of a novel class of elastase isozyme, human pancreatic elastase III, by cDNA and genomic gene cloning

By using porcine elastase I cDNA as a probe, we have isolated two different but closely related cDNAs encoding elastase-like proteases from a human pancreatic cDNA library. The amino acid sequences deduced from the cloned cDNA sequences showed 56-61% identity with those of both pancreatic elastases I and II, similar to the homology between elastases I and II. The active form of the elastase-like proteases appeared to be composed of 242 amino acids and preceded by a signal peptide and propeptide of 28 amino acids. Dot blot analysis of various tissue mRNAs demonstrated that the genes for the cloned cDNAs are expressed at a high level only in the pancreas. In addition, sequence analysis of the cloned genomic genes corresponding to one of the cDNAs showed that they are members of the elastase gene family. These results indicate that the two enzymes encoded by the cDNAs should be classified into a third class of elastase isozyme. Therefore, we designated them as human pancreatic elastases IIIA and IIIB. They strongly resembled cholesterol-binding pancreatic protease, suggesting that they may possess not only a digestive function but also function(s) related to cholesterol metabolism or transport in the intestine.     

Cloning and expression of human salivary-gland kallikrein in Escherichia coli.

CDNA clones for human kallikrein have been identified in a cDNA library constructed from mRNA of human salivary gland. The entire coding sequence for preprokallikrein and for the 5'- and 3'-untranslated regions were isolated by using a mixture of oligonucleotides corresponding to amino acids 51-56 of human urinary kallikrein and one oligonucleotide corresponding to amino acids 233-238 of human pancreatic kallikrein. The DNA sequence proved that, with the exception of two amino acid exchanges, kallikrein of the human salivary gland is identical with pancreatic kallikrein. Salivary gland and renal kallikrein was expressed in Escherichia coli from plasmid pKK223-3 under the control of the tac promoter. The protein was identified by Western-blot analysis and by demonstration of its specific proteolytic activity.