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.     

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.     

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.     

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.     

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.     

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 complete primary structure of phospholipase A2 from human pancreas

The complete amino acid sequence of phospholipase A2 (phosphatide 2-acylhydrolase, EC 3.1.1.4) from human pancreas was determined. The protein consists of a single polypeptide chain of 125 amino acids and has a molecular weight of 14003. The chain is cross-linked by seven disulfide bridges. The main fragmentation of the polypeptide chain was accomplished by digestion of the reduced and thialaminated derivative of the protein with clostripain, yielding three fragments. The largest fragment (residues 7-100) was further degraded both with staphylococcal proteinase and chymotrypsin. The sequence was determined by automated Edman degradation of the intact protein and of several large peptide fragments. Phospholipase A2 from human pancreas contains the same number of amino acids (125) as the enzyme from horse, while the enzymes from pig and ox contain 124 and 123 residues, respectively. The enzymes show a high degree of homology; human phospholipase differs from the other enzymes by substitutions of 26 (porcine), 28 (bovine) and 32 (equine) residues, respectively.     

Effect of poly(ADP-ribose) formation on DNA synthesis in chick-embryo-liver nuclei. Possible mechanism of template activation.

The complete amino acid sequence of bovine phospholipase A2 (EC 3.1.1.4) was determined. This enzyme has a molecular weight of 13 782 and consists of a single polypeptide chain of 123 amino acids cross-linked by seven disulfide bridges. The main fragmentation of the polypeptide chain was accomplished by digesting the reduced and thialaminated derivative of the protein with trypsin, staphylococcal protease and cyanogen bromide. A number of chymotryptic peptides were used for alignment and to obtain overlaps of at least two residues. The sequence of the peptides was determined by Edman degradation by means of direct phenylthiohydantoin identification in combination with identification as dansyl amino acids. Although 71% of all residues of phospholipase A2 from bovine, porcine and equine sources are conserved, bovine phospholipase A2 differs from the others by the total number of residues and by substitutions at 20 (porcine) and 33 (equine) positions.     

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.     

Crotalus adamanteus phospholipase A2-α: Subunit structure,NH2-terminal sequence,and homology with other phospholipases

Automated Edman degradation of reduced and carboxymethylated phospholipase A₂-α from Crotalus adamanteus venom revealed a single amino acid sequence extending 30 residues into the protein from the amino terminus. The singularity of the sequence and the yields of the phenylthiohydantoin amino acids thus obtained indicate that the subunits comprising the phospholipase dimer are identical. Further chemical evidence in support of subunit identity was obtained by cleavage of phospholipase A₂-α with cyanogen bromide. Compositional analysis of the protein revealed one residue of methionine per monomer and the sequence determination placed this amino acid at position 10 in the sequence of 133 amino acids. Cyanogen bromide cleavage of the protein, followed by reduction and carboxymethylation afforded the expected 2 fragments: an NH₂-terminal decapeptide (CNBr-1) and a larger COOH-terminal fragment of 123 residues (CNBr-II). Automated Edman degradation of the latter has extended the sequence analysis to 54 residues in the NH₂-terminal segment of the monomer chain. Comparison of this sequence with those derived for phospholipases from other snake venoms, from bee venom, and from porcine pancreas has revealed striking homologies in this region of the molecules. As expected on the basis of their phylogenetic classification, the phospholipases from the pit vipers C. adamanteus and Agkistrodon halys blomhoffii are more similar to one another in sequence than to the enzyme from the more distantly related viper, Bitis gabonica. Furthermore, the very close similarities in sequence observed among all of these phospholipases in regions corresponding to residues 24 through 53 in the C. adamanteus enzyme suggest that this segment of the polypeptide plays an important role in phospholipase function and probably constitutes part of the active site.     

Conformational changes expected in endogenous opioid peptides upon their interaction with acidic lipids

The amino terminal amino acid sequence of purified human angiotensinogen has been determined. The first 25 residues with the exception of number 14 were identified. The sequence of the first ten amino acids is that of angiotensin I. The sequence surrounding the renin cleavage site in this protein is Leu-¹⁰Val-¹¹Ile-¹²His-¹³. Thus, human renin must cleave a Leu-Val peptide bond in human angiotensinogen to release angiotensin I, rather than a Leu-Leu bond as reported for other species. The differences between the sequence of human angiotensinogen and that previously reported for a tetradecapeptide derived from equine and porcine angiotensinogen may be responsible for the known species specificity of renin.     

Human Pancreatic Procolipase Expressed in Insect Cells: Purification and Characterization

A cDNA clone encoding human pancreatic procolipase was incorporated into a recombinant baculovirus. Spodoptera frugiperda insect cells infected with the recombinant baculovirus secreted procolipase into the medium, which could be isolated in a single step by immunoaffinity chromatography. The highly purified protein reactivated human pancreatic lipase in a concentration-dependent fashion and was efficiently converted to colipase by limited trypsin digestion. This expression system is suitable for producing amounts of procolipase adequate for biophysical studies.     

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.     

Primary structure of equine pituitary prolactin

Equine prolactin was determined to be a single chain protein of 199 amino acid containing two tryptophan and six cysteine residues, as found in other mammalian prolactins. The primary sequence of equine prolactin was obtained by automated Edman analyses of S-carboxymethylated protein and proteolytic fragments of modified protein. Of the known prolactin sequences, equine prolactin shows closest homology with porcine (93%) and fin whale (87-91%) prolactins. Genetic mutations have produced changes in 17 of 199 residues of equine prolactin relative to its putative ancestral precursor. Since equine growth hormone has undergone alterations in 4 of 191 residues relative to this putative precursor protein, these results support the theory that prolactins are evolving at a faster rate than growth hormones. Consistent with the previously determined circular dichroic spectrum of equine prolactin, 60% of the protein is predicted to form alpha helices.     

The complete amino acids sequence of human complex-forming glycoprotein heterogeneous in charge (protein HC)

The complete amino acid sequence of human protein HC isolated from the urine of a single individual (JL) was determined. The polypeptide chain contained 181 residues, had a calculated molecular weight of 20,435 and contained 3 cysteine residues at positions 34, 70 and 167. An intrachain disulfide bridge was formed by residues 70 and 167. No variation of the amino acid sequence of protein HC was found and can therefore not explain the charge heterogeneity of protein HC in a given individual. The amino acid sequence of protein HC was almost identical to the one reported for human α₁-microglobulin but contained 14 additional residues.     

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.     

The amino acid sequence of porcine intestinal calcium-binding protein.

The complete amino acid sequence of the calcium-binding protein (CaBP) from pig intestinal mucosa has been determined: Ac-Ser-Ala-Gln-Lys-Ser-Pro-Ala-Glu-Leu-Lys-Ser-Ile-Phe-Glu-Lys-Tyr-Ala-Ala-Lys-Glu-Gly-Asp-Pro-Asn-Gln-Leu-Ser-Lys-Glu-Glu-Leu-Lys-Gln-Leu-Ile-Gln-Ala-Glu-Phe-Pro-Ser-Leu-Leu-Lys-Gly-Pro-Arg-Thr-Leu-Asp-Asp-Leu-Phe-Gln-Glu-Leu-Asp-Lys-Asn-Gly-Asn-Gly-Glu-Val-Ser-Phe-Glu-Glu-Phe-Gln-Val-Leu-Val-Lys-Lys-Ile-Ser-Gln-OH. The N-terminal octapeptide sequence was determined by mass spectrometric analysis by Morris and Dell. The first 45 residues of bovine CaBP differ only in six positions from the corresponding sequence of the porcine protein, except that the sequence starts in position two of the porcine sequence. The mammalian intestinal CaBP's belong to the troponin-C superfamily on the basis of an analysis by Barker and Dayhoff.     

The isolation and characterization of rabbit motilin precursor cDNA.

The cDNA sequence of rabbit motilin precursor has been determined. The predicted amino acid sequence indicates that the precursor consists of 133 amino acids and includes a 25 amino acid signal peptide followed by the 22 amino acid motilin sequence and an 86 amino acid motilin associated peptide (MAP). As in the human and porcine precursors, two lysine residues follow motilin in the rabbit sequence. Rabbit motilin shares 64% amino acid sequence identity with human and porcine motilin, and all amino acid substitutions represent conservative changes. Amino acid sequence alignments of the rabbit, human and porcine MAP sequences suggest three functional/structural motifs corresponding to a putative endoproteinase recognition site, a putative PEST site and a potential posttranslational processing recognition element.