Neurite Outgrowth-Stimulating Peptide Derived from Bovine κ-Casein

We found a novel peptide that stimulates neurite outgrowth in Neuro-2a mouse neuroblastoma cells, from the pepsin-pancreatin digest of bovine κ-casein. The amino acid sequence of this peptide is Phe–Leu–Pro–Tyr–Pro–Tyr (FLPYPY), corresponding to peptidic sequence 76–81 of bovine κ-casein. The neurite outgrowth-stimulating activity of FLPYPY was seen over 10^?9 M. On the other hand, FLPYP and FLPYPYY, which corresponded to sequences 76–80 and 76–82 of bovine κ-casein respectively, were ineffective.     

Expression of kappa-casein in normal and neoplastic rat mammary gland is under the control of prolactin

An 820-nucleotide-long cDNA clone for the kappa-casein (the casein micelle-stabilizing protein) from rat mammary gland was isolated, and its nucleotide sequence was determined. The deduced amino acid sequence from the nucleotide sequence revealed a signal peptide, 21 amino acids long, and a mature protein of 157 amino acids. The signal peptide of rat kappa-casein was highly homologous to that of the precursor to ovine kappa-casein. However, little homology was apparent when the mature kappa-casein protein sequences from ovine or bovine sources were compared with rat kappa-casein. The kappa-casein mRNA content of the mammary tissue was found to increase during its functional differentiation. Prolactin appears to modulate the production of kappa-casein mRNA. Mammary glands of virgin females had no detectable kappa-casein mRNA; however, a marked induction of kappa-casein mRNA was obtained by intravenous infusion of prolactin. Mammary carcinomas did not follow the same pattern. 7,12-Dimethylbenz[a]anthracene-induced mammary carcinomas had normally low levels of kappa-casein mRNA, but intravenous prolactin infusion increased the levels by 2-fold. The MTW9 mammary carcinoma that grows only in the presence of high levels of mammotropic hormones had kappa-casein mRNA content equivalent to that in 10-day lactating rat mammary gland. Continuous venous infusion of prolactin to MTW9 mammary carcinoma did not modify the kappa-casein mRNA levels. Nitrosomethylurea-induced mammary carcinomas had no detectable kappa-casein mRNA, and intravenous prolactin infusion was unable to induce it.     

A reinvestigation of the amino acid sequences of bovine, rabbit, monkey, and human myelin basic proteins

In order to determine whether bovine, rabbit, and monkey myelin basic proteins (BPs) have the sequence Gly-His or His-Gly at positions corresponding to bovine sequence 76-77, we isolated the tryptic peptides encompassing the sequence in question in these proteins and cleaved them into dipeptides with dipeptidyl aminopeptidase I (EC 3.4.14.1). Analysis by gas chromatography/mass spectrometry of the dipeptides released showed that in no case did His follow Gly or Gly precede His. The identification of peptides Ala-Gln and His-Gly (bovine BP) and Ser-His and Gly-Arg (rabbit and monkey BPs) established the His-Gly sequence. A similar sequence analysis of tryptic peptide (80-91) of human BP confirmed the sequence Thr-Gln-Asp-Glu-Asn-Pro (80-85).     

Identification of human milk kappa-casein on polyacrylamide gels by differential staining with Ethyl-Stains-all and chymosin sensitivity

Ethyl-Stains-all (ESA), a cationic carbocyanine dye that stains phosphorylated, sialylated, and unmodified proteins differentially, was used to stain a human casein fraction enriched for its kappa-casein-like characteristics. The staining properties and chymosin sensitivity of this fraction were compared with those of human milk and bovine casein proteins. Phosphorylated human and bovine beta caseins stained blue with ESA. The sialic acid-containing bovine kappa-casein stained blue-green. The human kappa-like fraction was enriched for a protein that stained blue-green with ESA. Both bovine kappa-casein and the human blue-green-staining protein were susceptible to chymosin digestion at lower concentrations of chymosin than that required for digestion of beta-caseins. In each case, following chymosin digestion, a green-staining peptide of lower molecular weight replaced the original protein and para-kappa-casein was formed. Identification of human kappa-casein on SDS-polyacrylamide gels was based on its differential staining with ESA and chymosin sensitivity with respect to beta-casein.     

Primary structure of the casein macropeptide of kappa casein of buffalo]

The complete amino acid sequence of Italian water buffalo (Bubalus arnee) caseinomacropeptide, the C-terminal fragment released from kappa-casein by chymosin, has been determined. It contains 64 amino acid residues including one phosphoserine and differs from its bovine (Bos taurus) B counterpart by 10 amino acid substitutions. The sequence of the last 11 amino acid residues of para-kappa-casein is also reported. In relation to the Ala148/Asp substitution which is responsible for the different electrophoretic behaviour of bovine kappa-caseins B and A, water buffalo kappa-casein is homologous to the bovine variant B. It is suggested that a variant Thr136-Ala148 might be the wild type of the Bos genus.     

Peptide substrates for chymosin (rennin). Interaction sites in kappa-casein-related sequences located outside the (103-108)-hexapeptide region that fits into the enzyme''s active-site cleft.

The role of individual amino acid residues in the 98-102 and 111-112 regions of bovine kappa-casein in its interaction with the milk-clotting enzyme chymosin (rennin) was investigated. to this end the tryptic 98-112 fragment of kappa-casein was modified in its N- and/or C-terminal part by chemical (guanidation, ethoxyformylation, repeated Edman degradation) and enzymic (carboxypeptidase) treatments. Further, use was made of short synthetic kappa-casein analogues in which His-102 had been replaced by Pro or Lys. All peptides and their derivatives were tested comparatively at various pH values for their ability to act as chymosin substrates via specific cleavage of the peptide bond at position 105-106. The results indicate that in the alternating 98-102 sequence (His-Pro-His-Pro-His) the His as well as the Pro residues contribute to the substrate activity with no predominant role of any one of these groups. Another interaction site is formed by the Lys residue at position 111 of the substrate. A model of the enzyme-substrate complex is proposed. Herein the 103-108 fragment of the substrate, to be accommodated within the enzyme's active-site cleft, is brought into position by electrostatic binding (via His-98, His-100, His-102 and Lys-111) near the entrance of the cleft. These interactions are strongly supported by Pro residues at positions 99, 101, 109 and 110 of the substrate, which act as stabilizers of the proper conformation of the substrate in the enzyme-substrate complex.     

The ostrich pituitary contains a major peptide homologous to mammalian chromogranin A(1-76)

A major peptide related to the NH2-terminal fragment (position 1 to 76) of mammalian chromogranin A was isolated from ostrich adenohypophyses following acid-acetone extraction. The complete amino acid sequence of the homogenous peptide was deduced following automatic Edman degradation of the native peptide as well as of CNBr-, tryptic- and Lysobacter-derived peptides. The 76 amino acid sequence is strikingly homologous to bovine (80.3% sequence identity), porcine (79.0%), human (79.0%) and rat (72.4%) corresponding sequences, but much less so to human chromogranin B (22.4%). As this peptide is followed in bovine, porcine and human structure by a pair of basic residues (Lys-Lys), it could conceivably be produced during maturation in secretory granules. Finally, its structure appears to contain two potential amphipathic helices joined by the single disulfide bridge present in all chromogranin A and B molecules.     

Peptide substrates for chymosin (rennin). Kinetic studies with bovine kappa-casein-(103-108)-hexapeptide analogues.

Kinetic parameters have been determined for the reaction between chymosin (EC 3.4.23.4) and synthetic peptide analogues of the sequence Leu-Ser-Phe-Met-Ala-Ile around the chymosin-sensitive Phe(105)-Met(106) bond of bovine kappa-casein. From the present and earlier results it is concluded that a minimum length of the molecular backbone with three amino acid units on both sides of the scissile bond is required to make the peptide a good substrate for the enzyme. In addition, hydrophobic side chains in the positions 103 and 108, and particularly the hydroxyl group of Ser-104 contribute to the effectiveness of the enzyme-substrate interactions. The substrate properties are markedly influenced by changes in the steric and/or polar character of the amino acid side chains in the positions 105 and 106.     

Structural and functional characterization of full-length heparin-binding growth associated molecule.

Heparin-binding growth-associated molecule (HB-GAM) was purified from adult bovine brain and chicken heart. The yield of HB-GAM is increased by 5- to 10-fold when 250 mM NaCl is added to the homogenization buffer, indicating that HB-GAM may exist as a complex with an insoluble component of the tissue. The complete amino acid sequence of the brain-derived HB-GAM was established by automated Edman degradation of the intact protein and chemically or enzymatically derived fragments. The mass of bovine HB-GAM as determined by plasma desorption time-of-flight mass spectrometry is 15,291 mass units, which compares favorably with the calculated mass of 15,289 based on the amino acid sequence. Therefore, HB-GAM has not undergone any major post-translational modifications other than cleavage of the signal peptide. These results indicate that previous amino acid sequence analysis of this protein was carried out using truncated HB-GAM. Full-length HB-GAM is not a mitogen for Balb/3T3 clone A31, Balb MK, NRK, or human umbilical vein endothelial cells. HB-GAM does, however, have adhesive properties and neurite extension activity for chick embryo cerebral cortical derived neurons when presented to these cells as a substrate. HB-GAM had little neurite extension activity when presented as a soluble factor.     

Nucleotide sequences of bovine alpha S1- and kappa-casein cDNAs

The nucleotide sequences corresponding to bovine alpha S1- and kappa-casein mRNAs are presented. An unusual alpha S1-casein cDNA has been characterised whose 5' end commences upstream from its putative TATA box. The alpha S1-casein mRNA is compared to rat alpha-casein mRNA and two components of divergence are identified. Firstly, the two sequences have diverged at a high point mutation rate and the rate of amino acid replacement by this mechanism is at least as great as the rate of divergence of any other part of the mRNAs. Secondly, the protein coding sequence has been subjected to several insertion/deletion events, one of which may be an example of exon shuffling . The kappa-casein mRNA sequence verifies the proposition that it has arisen from a different ancestral gene to the other caseins.     

Cloning of the authentic bovine gene encoding pepsinogen a and its expression in microbial cells

Bovine pepsin is the second major proteolytic activity of rennet obtained from young calves and is the main protease when it is extracted from adult animals, and it is well recognized that the proteolytic specificity of this enzyme improves the sensory properties of cheese during maturation. Pepsin is synthesized as an inactive precursor, pepsinogen, which is autocatalytically activated at the pH of calf abomasum. A cDNA coding for bovine pepsin was assembled by fusing the cDNA fragments from two different bovine expressed sequence tag libraries to synthetic DNA sequences based on the previously described N-terminal sequence of pepsinogen. The sequence of this cDNA clearly differs from the previously described partial bovine pepsinogen sequences, which actually are rabbit pepsinogen sequences. By cloning this cDNA in different vectors we produced functional bovine pepsinogen in Escherichia coli and Saccharomyces cerevisiae. The recombinant pepsinogen is activated by low pH, and the resulting mature pepsin has milk-clotting activity. Moreover, the mature enzyme generates digestion profiles with alpha-, beta-, or kappa-casein indistinguishable from those obtained with a natural pepsin preparation. The potential applications of this recombinant enzyme include cheese making and bioactive peptide production. One remarkable advantage of the recombinant enzyme for food applications is that there is no risk of transmission of bovine spongiform encephalopathy.     

Specificity of hydrolysis of bovine kappa-casein by cell envelope-associated proteinases from Lactococcus lactis strains.

The cell envelope-associated proteinases from Lactococcus lactis subsp. cremoris H2 (a PI-type proteinase-producing strain) and SK11 (a PIII-type proteinase-producing strain) both actively hydrolyze the kappa-casein component of bovine milk but with significant differences in the specificity of peptide bond hydrolysis. The peptide bonds Ala-23-Lys-24, Leu-32-Ser-33, Ala-71-Gln-72, Leu-79-Ser-80, Met-95-Ala-96, and Met-106-Ala-107 were cleaved by both proteinase types, although the relative rates of hydrolysis at some of these sites were quite different for the two proteinases. Small histidine-rich peptides were formed as early products of the action of the cell envelope-associated proteinases on kappa-casein, implicating this casein as a possible significant source of histidine, which is essential for starter growth. The major difference between the two proteinase types in their action on kappa-casein was in their ability to cleave bonds near the C-terminal end of the molecule. The bond Asn-160-Thr-161 and, to a lesser extent, the bond Glu-151-Val-152 were very rapidly cleaved by the PIII-type proteinase, whereas hydrolysis of these bonds by the PI-type proteinase was barely detectable (even after 24 h of digestion). Differential hydrolysis of kappa-casein at these sites by the two different proteinase types resulted in the formation of distinctive, high-M(r) products detectable by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Detection of the cholera toxin-binding activity of kappa-casein macropeptide and optimization of its production by the response surface methodology

The cholera toxin (CT)-binding activity of purified kappa-casein macropeptide (CMP) from bovine kappa-casein was detected. In addition, a statistical model was developed to optimize the production of CMP. CMP was prepared by chymosin hydrolysis of kappa-casein and a subsequent 3% trichloroacetic acid treatment. CMP was further fractionated in an ion-exchange column by FPLC. CT binding activity was eluted at 0.18 M NaCl and was a single 8.9 kDa peptide without tyrosine and arginine residues. The CT binding activity was rapidly lost by a carbohydrase treatment. The conditions for CMP production with chymosin were optimized by using the response surface methodology (RSM). The estimated optimum levels of the factors were as follows: reaction temperature, 38.5 degrees C; pH, 6.44; and time, 35.9 min. A validation experiment was performed in which CMP was prepared under the predicted parameters, and it was ascertained that the estimated optimum conditions gave better production of CMP than any other conditions.     

Intermolecular cross-linking and stereospecific molecular packing in type I collagen fibrils of the periodontal ligament

A trypsin digest of denatured NaB3H4-reduced native bovine periodontal ligament was prepared and fractionated by gel filtration and cellulose ion-exchange column chromatography. Prior to trypsin digestion, a complete acid hydrolysate was subjected to analyses for nonreducible stable and reducible intermolecular cross-links. Minute amounts of the former and significant amounts of the reduced cross-links dihydroxylysinonorleucine (1.1 mol/mol of collagen), hydroxylysinonorleucine (0.9 mol/mol of collagen), and histidinohydroxymerodesmosine (0.6 mol/mol of collagen) were found. The covalent intermolecular cross-linked two-chained peptides that were isolated were subjected to amino acid and sequence analyses. The structures for the different two-chained linked peptides were alpha 1CB4-5(76-90)[Hyl-87] X alpha 1CB6-(993-22c)[Lysald-16c], alpha 1CB4-5(76-90)[Hyl-87] X alpha 1CB6(993-22c)[Hylald-16c], alpha 2CB4(76-90)[Hyl-87] X alpha 1CB6(993-22c)[Lysald-16c], and alpha 2CB4(76-90)[Hyl-87] X alpha 1CB6(993-22c)[Hylald-16c]. The cross-link in each peptide was glycosylated. This is the first characterization by sequence analysis of a cross-link involving Hyl-87 in an alpha 2 chain in collagen. A stoichiometric conversion of residue 16c aldehyde to an intermolecular cross-link in each of the COOH-terminal nonhelical peptide regions of both alpha 1 chains in a molecule of type I collagen was found. The ratio of alpha 1 to alpha 2 intermolecularly cross-linked chains involved was 3.3:1, indicating a stereospecific three-dimensional molecular packing of type I collagen molecules in bovine periodontal ligament.     

Location of the intermolecular cross-links in bovine dentin collagen,solubilization with trypsin and isolation of cross-link peptides containing dihydroxylysinonorleucine and pyridinoline

[³H]NaBH₄ reduced bovine dentin collagen was denatured at 60°C for 1 hr and then digested with trypsin. The digest was still substantially insoluble suspension, but it was found that 99% of dentin collagen can be solubilized if the digest was heated again at 60°C for 15 min. Two cross-linked tryptic peptides were isolated from this digest by sequential chromatographies on Sephadex G50, phosphocellulose and DEAE-cellulose column. One isolated peptide was characterized as a 59 residue cross-linked peptide including one residue of dihydroxylysinonorleucine and the other was 103 residue including one residue of pyridinoline. The amino acid compositions were consistent with the identification of the 59 residue peptide as the sequence in α1-CB4-5 (76–90) linked to the sequence in α1-CB6 (990-23^c), and the 103 residue peptide as the sequence 76–90 linked to two of the sequence 990-23^c. These results strongly support the previously proposed precursor-product relationship between dihydroxylysinonorleucine and pyridinoline.     

Specificity of milk-clotting enzymes towards bovine kappa-casein

Limited proteolysis of bovine kappa-casein has been investigated with porcine pepsin A and C, and with the 2 microbial proteinases Mucor miehei proteinase and Endothia parasitica proteinase. The liberated C-terminal glycomacropeptide of kappa-casein was isolated after precipitation in 3% trichloroacetic acid followed by high-performance gel-permeation chromatography on a TSK G3000 SW column. From amino acid analyses and N-terminal sequencing of the liberated peptide it is concluded that porcine pepsin A, C and Mucor miehei proteinase cleave the same bond as chymosin: Phe-105-Met-106 whereas Endothia parasitica proteinase cleaves the bond Ser-104-Phe-105.     

Cloned transgenic cattle produce milk with higher levels of beta-casein and kappa-casein

To enhance milk composition and milk processing efficiency by increasing the casein concentration in milk, we have introduced additional copies of the genes encoding bovine beta- and kappa-casein (CSN2 and CSN3, respectively) into female bovine fibroblasts. Nuclear transfer with four independent donor cell lines resulted in the production of 11 transgenic calves. The analysis of hormonally induced milk showed substantial expression and secretion of the transgene-derived caseins into milk. Nine cows, representing two high-expressing lines, produced milk with an 8-20% increase in beta-casein, a twofold increase in kappa-casein levels, and a markedly altered kappa-casein to total casein ratio. These results show that it is feasible to substantially alter a major component of milk in high producing dairy cows by a transgenic approach and thus to improve the functional properties of dairy milk.     

Pyridinoline, a non-reducible crosslink of collagen. Quantitative determination, distribution, and isolation of a crosslinked peptide

Pyridinoline is a crosslink compound isolated from bovine Achilles tendon collagen. It is a 3-hydroxypyridinium derivative with three amino and three carboxyl groups (Fujimoto, D., Akiba, K., & Nakamura, N. (1977) Biochem. Biophys. Res. Commun. 76, 1124-1129). The contents of pyridinoline in collagens from various sources were determined. The pyridinoline content of bovine Achilles tendon was 0.16 residue per 1,000 residues and that of rat Achilles tendon collagen was 0.017 residue per 1,000 residues. Besides Achilles tendon collagens, pyridinoline was found in collagens from costal cartilage, rib and femoral bone of rat. It was not found in collagens from the tail tendon and skin of rat. A crosslinked, triple-chained peptide containing pyridinoline was isolated from bovine Achilles tendon collagen after digestion with pronase. Its amino acid composition suggests that the peptide may be involved in an intermolecular crosslink among a carboxyterminal sequence, a sequence near the aminoterminus and a sequence in the helical region.     

Glycosylated kappa-caseins and the sizes of bovine casein micelles. Analysis of the different forms of kappa-casein

Fast protein liquid chromatography (FPLC) of the kappa-casein from bovine casein micelles of different sizes is used to demonstrate that the proportions of glycosylated and non-glycosylated forms of kappa-casein do not vary with micellar size. The results suggest that glycosylated kappa-casein is distributed similarly to unglycosylated kappa-casein within the micellar structure.