Tat'yana G. Kanyshkova

Lactoferrin and its biological functions.

Lactoferrin, a component of mammalian milk, is a member of the transferrin family. These glycoproteins transfer Fe3+ ions. Lactoferrin is a unique polyfunctional protein that influences cell proliferation and differentiation. It can regulate granulopoiesis and DNA synthesis in some cells. Lactoferrin inhibits prostaglandin synthesis in human milk macrophages and activates the nonspecific immune response by stimulating phagocytosis and complement. It can interact with DNA, RNA, proteins, polysaccharides, heparin-like polyanions, etc.; in some of its effects, lactoferrin is found in complexes with ligands. It was recently demonstrated that lactoferrin also possesses ribonuclease activity and is a transcription factor. The list of known biological activities of lactoferrin is constantly increasing. This review analyzes possible mechanisms of its polyfunctionality.

Hydrolysis of myelin basic protein by polyclonal catalytic IgGs from the sera of patients with multiple sclerosis

Various catalytic antibodies or abzymes have been detected recently in the sera of patients with several autoimmune pathologies, where their presence is most probably associated with autoimmunization. Recently we have shown that DNase, RNase, and polysaccharide‐hydrolyzing activities are associated with IgGs from the sera of patients with multiple sclerosis (MS). Here we present evidence demonstrating that highly purified MS IgGs (but not Igs from the sera of healthy individuals) catalyze specifically hydrolysis of human myelin basic protein (hMBP). In contrast to many known proteases, IgGs do not hydrolyze many other different proteins. Specific inhibitors of acidic and thiol proteases have no remarkable effect on proteolytic activity of IgGs. However, specific inhibitor of serine (PMSF, AEBSF, and benzamidin) and metal‐dependent (EDTA) proteases significantly inhibit activity of proteolytic abzymes. Interestingly, the ratio of serine‐like and metal‐dependent activities of MS IgGs varied very much from patient to patient. The findings speak in favor of the generation by the immune systems of individual MS patients of a variety of polyclonal anti‐MBP IgGs with different catalytic properties.

Catalytic heterogeneity of polyclonal DNA-hydrolyzing antibodies from the sera of patients with multiple sclerosis

Various catalytic antibodies or abzymes have been detected recently in the sera of patients with several autoimmune pathologies, where their presence is most probably associated with autoimmunization. Recently we have shown that DNase activity is associated with IgGs from the sera of patients with multiple sclerosis (MS) but not with those from the sera of normal humans. Here we present evidence showing that MS IgG, its F(ab) fragments, and separated L-chains catalyze DNA hydrolysis. The properties of the DNase activity of these polyclonal IgGs distinguish them from other known human DNases. In addition, their specific activities with different oligonucleotide substrates and the range of optimal pHs, apparent K(M) values and substrate specificities varied widely for different patients. The findings speak in favor of the generation by the immune systems of individual patients of a variety of polyclonal catalytic IgG pools, from relatively small to extremely large ones.

DNA-hydrolyzing activity of the light chain of IgG antibodies from milk of healthy human mothers

Various catalytically active antibodies or abzymes have been detected recently in the sera of patients with several autoimmune pathologies, where their presence is most probably associated with autoimmunization. Normal humans are generally considered to have no abzymes, since no obvious immunizing factors are present. Recently we have shown that IgG (its Fab and F(ab)2 fragments) from the milk of normal humans possesses DNase activity. Here we demonstrate for the first time that the light chain of IgG catalyzes the reaction of DNA hydrolysis. These findings speak in favor of the generation of abzymes in the tissue of healthy mothers, and since a mothers breast milk protects her infant from infections until the immune system is developed, they raise the possibility that these abzymes may contribute to this protective role.

Secretory immunoglobulin A from healthy human mothers' milk catalyzes nucleic acid hydrolysis.

The human milk secretory immune system is known to be the first line of protection for the newborn infant against various pathogens. Secretory IgA (sIgA), the typical immunoglobulin found in secretions, can fight infections through many mechanisms. Using different methods, we have shown that sIgA from the milk of healthy women possesses DNAse and RNAse activities. The catalytic center is localized in the light chain of catalytic sIgA, while the DNA-binding center is predominantly formed by its heavy chain. The enzymic properties and substrate specificity of catalytic sIgA distinguish it from other known DNases and RNases. It is reasonable to assume, that the milk DNA- and RNA-hydrolyzing antibodies are capable not only of neutralizing viral and bacterial nucleic acids by binding these antigens as well as by hydrolyzing them. The DNA-hydrolyzing activity of Abs raises the possibility that these catalytic Abs may provide protective functions for the newborn through the hydrolysis of viral and bacterial nucleic acids.

Amylolytic activity of IgM and IgG antibodies from patients with multiple sclerosis

IgG and IgM antibodies from the sera of patients with multiple sclerosis (MS) were found to possess amylolytic activity hydrolyzing alpha-(1-->4)-glucosyl linkages of maltooligosaccharides, glycogen, and several artificial substrates. Individual IgM fractions isolated from 54 analyzed patients with the clinically definite diagnoses of MS had approximately three orders of magnitude higher specific amylolytic activity than that for healthy donors, whereas IgG from only a few patients had high amylolytic activity. Strict criteria were used to prove that the amylolytic activity of IgMs and IgGs is their intrinsic property and is not due to any enzyme contamination. Fab fragments produced from IgM and IgG fractions of the MS patients displayed the same amylolytic activity. IgMs from various patients demonstrated different modes of action in hydrolyzing maltooligosaccharides.

Amylolytic activity of IgG and sIgA immunoglobulins from human milk.

BACKGROUND New natural amylolytic abzymes (Abs) for catalytically active antibodies from human milk have been identified and investigated. METHODS The amylolytic activity of human milk autoantibodies was studied by TLC and HPLC techniques analyzing the hydrolysis of maltooligosaccharides with different degrees of polymerization and of 4-nitrophenyl 4,6-O-ethylidene-alpha-D-maltoheptaoside (EPS). IgG and sIgA fractions were isolated from human milk by affinity chromatography. After SDS-PAGE preparation of native IgG and sIgA and their renaturation, the amylolytic activity was in-gel assayed. RESULTS All electrophoretically homogeneous preparations of IgG and its Fab fragments as well as sIgA antibodies possessed alpha-amylolytic activity. The specific activities of these catalytic antibodies varied in the range from 1.83 up to 3.33 kat/kg, which is about one order of magnitude higher than that for IgGs from the sera of cancer patients. IgG and sIgA fractions showed Michaelis constants for hydrolysis of 4-nitrophenyl 4,6-O-ethylidene-alpha-D-maltoheptaoside in the range of 10(-4) M/l. Fractions of autoantibodies from different donors exhibited different modes of action in hydrolysis of maltooligosaccharides, maltose and p-nitrophenyl-alpha-D-glucopyranose. CONCLUSIONS IgG antibodies, their Fab fragments, and sIgA fractions isolated from human milk of healthy women possessed amylolytic activity in the hydrolysis of maltooligosaccharides and several artificial substrates.

Lactoferrin Is the Major Deoxyribonuclease of Human Milk

Lactoferrin is the major iron-transferring protein of human barrier fluids such as blood and milk. It is a polyfunctional protein capable of binding DNA exposed on the surface of various cells. Electrophoretically homogenous lactoferrin was prepared by sequential chromatography of human milk proteins on DEAE-cellulose, heparin-Sepharose, and Sepharose containing immobilized anti-lactoferrin antibodies. By subsequent chromatography on Blue Sepharose the resulting lactoferrin was fractionated into several subfractions with different affinity for the sorbent, and this was associated with separation of additional lactoferrin peaks with DNase activity from the main peak. By various techniques, in particular, by in situ testing the DNase activity of lactoferrin in a DNA-containing gel after SDS-electrophoresis, hydrolysis of DNA was for the first time shown to be an intrinsic property of lactoferrin. The substrate specificity of lactoferrin in hydrolysis of DNA was different from specificities of known human DNases. Hydrolysis of DNA was activated by bivalent metal ions and also by ATP and NAD. Unlike the main fraction of lactoferrin with the highest affinity for Blue Sepharose, all protein subfractions with DNase activity were cytotoxic and suppressed growth of human and mouse tumor cell lines.

HUMAN MILK LACTOFERRIN BINDS ATP AND DISSOCIATES INTO MONOMERS

The physiological role of lactoferrin (LF) is still unclear, but it has been suggested to be responsible for primary defence against microbial infections. Many different unique functions have been attributed to LF, including DNA and RNA binding, and transport into the nucleus, where LF binds to specific DNA sequences and activates transcription. Here we present evidence that in addition to the above (and below) mentioned functions LF binds ATP with a stoichiometry of 1 mole of nucleotide per mole of the protein and a Kd=0.3 mM. The ATP‐binding site is localized in the C‐terminal domain of LF, in contrast to the antibacterial and polyanion‐binding sites, which are located in the N‐terminal domain. Binding of ATP by LF leads to dissociation of its oligomeric forms and to a change of the proteins interaction with polysaccharides, DNA and proteins.