Lone K. Rasmussen

Bovine milk procathepsin D and cathepsin D: coagulation and milk protein degradation.

Cathepsin D is an indigenous aspartic proteinase in bovine milk. By competitive enzyme-linked immunosorbent assay the amount of immunoreactive cathepsin D and procathepsin D in bovine skim milk was estimated to be 0.4 microgram/ml. Immunoreactive cathepsin D purified from whey consisted of a small fraction of mature cathepsin D, but the major form was the proenzyme procathepsin D. A preparation of bovine milk procathepsin D was, like mature cathepsin D, able to degrade purified alpha s1-, alpha s2-, beta- and kappa-casein and alpha-lactalbumin, while beta-lactoglobulin was resistant to cleavage. The cleavage sites in these proteins were determined and compared with those of chymosin. Cathepsin D was capable of generating the alpha s1-I, beta-I, beta-II and beta-III fragments originally described from the action of chymosin on the respective caseins, and these fragments were subjected to further proteolysis. Cathepsin D was also able to liberate the caseinomacropeptide from purified kappa-casein, and to coagulate bovine skim milk. This demonstrated that milk contains an indigenous coagulation enzyme present mainly in the whey fraction.

Identification of glutamine and lysine residues in Alzheimer amyloid βA4 peptide responsible for transglutaminase-catalysed homopolymerization and cross-linking to α2M receptor

The β‐amyloid peptide (βA4), derived from a larger amyloid precursor protein, is the principal component of senile plaques in Alzheimers disease. Here we report that the full‐length (1–40) synthetic βA4 peptide, containing one glutamine and two lysine residues, is able to form homopolymers in a transglutaminase‐mediated reaction. Moreover, transglutaminase catalysed the formation of heteropolymers in reactions of βA4 with α2M receptor, a constituent of amyloid plaques, and with extracellular matrix proteins. Incorporation of site‐specific probes followed by enzymatic digestion and sequencing of tracer‐containing fractions demonstrated that both Lys16, Lys28 and Gin15 in βA4 were susceptible to cross‐linking by transglutaminase.

Very low density lipoprotein receptor from mammary gland and mammary epithelial cell lines binds and mediates endocytosis of M(r) 40,000 receptor associated protein.

We here report that the M r 40,000 receptor associated protein (RAP), previously found to bind to α2‐macroglobulin receptor/low density lipoprotein receptor related protein (α2MR/LRP) and glycoprotein 330 (gp330), binds to an M r, 105,000 membrane protein from bovine mammary gland, human mamma tumors and mammary epithelial cell lines. We have purified this protein from bovine and human sources. N‐terminal amino acid sequencing and immunoblotting analyses showed that the protein was identical or closely related to very low density lipoprotein receptor (VLDL‐R). Experiments with the human mamma carcinoma cell line MCF‐7 showed that this receptor was able to mediate an efficient endocytosis of RAP. These novel findings strongly suggest that RAP functions as a modulator of ligand binding to VLDL‐R, similarly to α2MR/LRP and gp330.

The plasminogen activation system in bovine milk: Differential localization of tissue-type plasminogen activator and urokinase in milk fractions is caused by binding to casein and urokinase receptor

We have analyzed the occurrence of components of the plasminogen activation system in bovine milk. Zymographic analyses showed that tissue-type plasminogen activator (t-PA) occurred in association with casein micelles, partially as a complex with type-1 plasminogen activator inhibitor (PAI-1), whereas urokinase-type plasminogen activator (u-PA) was confined to milk leukocytes. Whey contained a component with a plasminogen dependent proteolytic activity which was shown to be plasma prekallikrein (PPK). The u-PA in the milk leukocytes was shown to be bound to urokinase receptor (u-PAR). A purification to near-homogeneity of the bovine u-PAR was undertaken. Investigating the novel t-PA binding to casein micelles by ligand blotting and Sepharose immobilized casein, multimeric forms of kappa-casein and dimeric alpha s2-casein were identified as t-PA binding components. The kappa-casein gene and the fibrinogen gene are believed to have evolved from a common ancestor. Thus, the recent finding that casein enhances t-PA catalyzed plasminogen activation (Marcus, G., Hitt, S., Harvey, S.R. and Tritsch, G.L. (1993) Fibrinolysis 7, 229-236), and the observed t-PA/casein binding suggests that the casein micelle, which also contains plasminogen, may serve as a matrix for t-PA-catalyzed plasminogen activation in milk.

Disulphide-linked caseins and casein micelles

Abstract Here we report the disulphide arrangement as well as the multimeric structure of α s2 - and κ -casein in various species. Furthermore, the structure of the casein micelle based on liquid-state and solid-state NMR studies is discussed.

Localization of two interchain disulfide bridges in dimers of bovine αs2‐casein

Carboxymethylation of bovine skimmed milk with 14C-labelled iodoacetic acid followed by purification of the alpha s2-casein dimer showed that all four cysteine residues in the protein are engaged in disulfide linkages. Mass spectrometry and sequence analysis of cystine-containing tryptic peptides revealed the presence of two interchain disulfide bridges in the protein. Sequence analysis of disulfide-linked peptides resulting from an enzymatic cleavage between the bridges demonstrated that the individual chains in the dimers are either aligned in an antiparallel or a parallel orientation. The identity of some of the disulfide-linked peptides was further verified by performic acid oxidation followed by sequence analysis of the resulting peptides.

Disulphide arrangement in bovine caseins: localization of intrachain disulphide bridges in monomers of κ- and αs2-casein from bovine milk

Naturally occurring monomeric kappa-casein and alpha s2-casein in bovine milk were purified by ion-exchange chromatography in order to localize potential intrachain disulphide bridges. Enzymic cleavage of the proteins followed by mass spectrometry and amino acid sequence analysis of cystine-containing peptides revealed the presence of an intrachain disulphide bond in both proteins.

The Clostridium ramosum IgA Proteinase Represents a Novel Type of Metalloendopeptidase

Clostridium ramosum is part of the normal flora in the human intestine. Some strains produce an IgA proteinase that specifically cleaves human IgA1 and the IgA2m(1) allotype. This prolylendopeptidase was purified from a broth culture supernatant, and N-terminal sequences of the native protein and tryptic fragments thereof were determined. A fragment of the igagene encoding the IgA proteinase was isolated using degenerate primers in PCR, and the complete gene was obtained by inverse PCR. The identity of the iga gene was confirmed by heterologous expression inEscherichia coli. The deduced amino acid sequence indicated a signal peptide of 30 residues and a secreted proteinase of 133,828 Da. A typical Gram-positive cell wall anchor motif was identified in the C terminus. The presence of a putative zinc-binding motif His-Glu-Phe-Gly-His together with inhibition studies indicate that the proteinase belongs to the zinc-dependent metalloproteinases. However, the sequence of the C. ramosumIgA proteinase shows no overall similarity to other proteins except for significant identity around the zinc-binding motif with family M6 of metalloendopeptidases, and the unique sequence of the IgA proteinase in this area presumably establishes a new subfamily. The GC percentage of the iga gene is significantly higher than that for the entire genome of C. ramosum, suggesting that the gene was acquired recently in evolution.

Plasminogen activation system in human milk.

BACKGROUND Plasmin is the major endogenous protease present in milk. The level of plasmin activity is controlled by the availability of the precursor plasminogen and by the levels of plasminogen activators and inhibitors. Recently, a differential distribution of tissue-type plasminogen activator (t-PA) and urokinase-type plasminogen activator (u-PA) has been demonstrated in bovine milk. To assess whether this distribution pattern is a general feature, the occurrence of components of the plasminogen activation system in different fractions of human milk was investigated. METHODS Milk samples were separated into the following fractions; milk fat, skim milk, and milk cells by centrifugation. The different fractions were detected for the presence of plasminogen and plasminogen activators by immunoblotting and zymography. The distribution of t-PA and u-PA was investigated by ligand binding analysis. t-PA-catalyzed plasminogen activation was examined by a coupled chromogenic assay. RESULTS A differential distribution of plasminogen, t-PA, and u-PA was found. Casein micelles were found to exhibit t-PA and plasminogen binding activity, whereas the u-PA receptor was identified as the u-PA binding component in the cell fraction. Furthermore, human casein enhanced t-PA-catalyzed plasminogen activation, comparable to the enhancing effect obtained with fibrinogen fragments. CONCLUSION The finding of a differential distribution of u-PA and t-PA in milk suggests that the two activators may have different physiological functions, which involve protection against invading microorganisms and maintenance of patency and fluidity in the ducts of mammary gland, respectively.

Purification of disulphide-linked αs2- and κ-casein from bovine milk

: Naturally occurring disulphide-linked alpha s2- and kappa-casein in bovine milk were purified by gel chromatography on a column of Sepharose CL-6B. Four fractions (A-D) were obtained by elution with ammonium acetate-urea buffer. Fractions A and B, identified by SDS gel electrophoresis and amino acid sequence analysis, corresponded to disulphide-linked kappa-casein and alpha s2-casein respectively. Fraction C consisted of a mixture of alpha s1-, alpha s2-, and beta-casein. Separation of fraction C into its components was achieved by reversed-phase HPLC. The stability of the disulphide bridges in alpha s2- and kappa-casein was shown to differ with respect to reducing agents (dithioerythritol and 2-mercaptoethanol).