Maria Janusz

ISOLATION AND CHARACTERIZATION OF A PROLINE-RICH POLYPEPTIDE FROM OVINE COLOSTRUM

Department of Immunochemistry, Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, 4 Chalubinski Str., 50 368 Wroclaw, Poland, and Czechoslovak Academy of Sciences, Institute of Organic Chemistry and Biochemistry, I6 610 &aha 6, Czechoslovakia

Colostrinin, a Polypeptide Isolated From Early Milk, Facilitates Learning and Memory in Rats

Initial observations in humans indicated that colostrinin, a complex of polypeptides derived from the colostrum of sheep, facilitates cognitive functioning in patients with Alzheimers disease. Its effect on learning and memory in more controlled settings as well as the specificity of these effects were, however, unknown. The present experiments evaluated the effects of colostrinin on spatial learning (Morris water maze) and incidental memory (habituation test) in male Wistar rats of two age groups. Colostrinin, at a dose of 4 microg/rat IP, facilitated acquisition of spatial learning of 13- (aged) but not 3-month-old (young) rats. At the same dose, it improved incidental learning in aged rats, while the dose of 20 microg/rat attenuated it. Colostrinin did not change locomotor activity of rats. Taken together, the present findings indicate that colostrinin may have some beneficial effects on cognitive functioning, particularly in aged subjects. Given the fact that colostrum is the first nutritive agent of neonates, it might be speculated that its peptides may facilitate the early postnatal development of the cerebral neurons and their plasticity.

Towards an understanding of biological role of colostrinin peptides.

The aim of this study was to elucidate the structure and possible function of colostrinin, also known as a proline rich polypeptide (PRP). The molecular weight of colostrinin was originally determined by gel filtration to be 17,200 daltons. In the presence of guanidinum chloride, however, the molecular weight was found to be about 6,000 daltons. Further studies utilizing high-performance liquid chromatography (HPLC) and mass spectroscopy revealed that colostrinin is a complex consisting of many low molecular-weight polypeptides. A total of 32 peptides were isolated from the original colostrinin preparation by HPLC and subjected to the N-terminal sequence analysis. The results of sequence analysis revealed significant homology of the peptides to three protein precursors: annexin, β-casein, and a hypothetical β-casein homolog. In addition, the sequence of several peptides showed no significant homology to any specific protein in the current GenBank database. The synthetic peptides of various lengths representing the N-terminal sequence of the colostrinin peptides were made to study some biological effects. Here we report that colostrinin and some of its component peptides are potent inducers of leukocyte proliferation and of certain cytokines. Also, a series of monospecific antibodies were produced in rabbits against the synthetic peptides. The antibodies were used to study the kinetic of antigen reduction in colostrum and mature milk following lambing. A threefold decrease was common for most antigens studied over the period of 72 h. Based on the results of these studies we postulate that colostrinin represents a diverse group of peptides produced in the mammary gland of mammals for the development of the optimal physiologic responses in offspring. Also, it is hoped that the beneficial use of colostrinin in Alzheimer’s Disease (AD), recently reported elsewhere, will revive interest in its clinical application for treatment and/or prophylaxis of many age-related disorders.

Immunoregulatory properties of synthetic peptides, fragments of a proline-rich polypeptide (PRP) from ovine colostrum

It has been previously found that a proline-rich polypeptide (PRP) isolated from ovine colostrum has a regulatory effect on the immune response. A nonapeptide fragment Val-Glu-Ser-Tyr-Val-Pro-Leu-Phe-Pro was isolated from the chymotryptic digest of PRP. The nonapeptide showed biological activity similar to PRP. The determined amino acid sequence was now confirmed by synthesis. Synthetic nonapeptide as well as its C-terminal hexapeptide, Tyr-Val-Pro-Leu-Phe-Pro, showed biological activity similar to PRP and the nonapeptide obtained from PRP.

Immunologically active nonapeptide fragment of a proline-rich polypeptide from ovine colostrum: amino acid sequence and immunoregulatory properties.

It has been previously found that a proline-rich polypeptide (PRP) isolated from ovine colostrum has a regulatory effect on the immune response. To study the relationship between the structure of PRP and its immunomodulatory properties, the polypeptide was digested by chymotrypsin. Products of the proteolysis were separated by gel filtration and three fractions were obtained: PRP-1, PRP-2 and PRP-3. The activity of the fractions was compared with the activity of the untreated PRP. It was found that PRP-1 was inactive, whereas PRP-2 and PRP-3 showed an activity in the regulation of the immune response assayed by measurement of PFC, and by studying effects on delayed hypersensitivity, formation of autologous rosette-forming cell, and sensitivity of thymocytes to hydrocortisone. The activity of PRP-2 and PRP-3 was comparable to the activity of PRP. The PRP-3 fraction of low mol. wt was further purified and a pure nonapeptide of mol. wt 1000 (PRP-3b) was isolated. The amino acid sequence of PRP-3b was: Val--Glu--Ser--Tyr--Val--Pro--Leu--Phe--Pro. The nonapeptide showed the full spectrum of biological activities of PRP. Comparison of terminal amino acid suggested that PRP-3b was neither the NH2- nor the COOH-terminal fragment of PRP. The amino acid sequence of the nonapeptide indicated that PRP-3b is different from other known immunomodulators.

Immunoglobulins of colostrum—III: Comparative studies of ovine serum and colostral IgG1 and IgG2

Abstract Comparative studies of physico-chemical properties of ovine serum and colostral IgG1 and IgG2 are described. Hydrodynamic properties, amino acid compositions, carbohydrate contents, and ORD and CD spectra were compared. Colostral immunoglobulins showed higher content of β-structure in comparison with serum immunoglobulins. Different amino acid compositions of serum and colostral immunoglobulins, especially in case of IgG2 was shown. A polypeptide with high content of proline and glutamic acid residues accompanying colostral IgG2 was isolated. The results obtained are suggesting that ovine colostral IgG2 is locally synthesized in mammary gland, wherease colostral IgG1 might be a mixed population of molecules locally synthesized and transferred from the serum.

A proline-rich polypeptide complex and its nonapeptide fragment inhibit nitric oxide production induced in mice.

A proline-rich polypeptide complex (PRP) isolated from ovine colostrum shows immunoregulatory and procognitive activities. It shows beneficial effects in Alzheimers disease (AD) patients when orally administered in the form of tablets called Colostrinin. The mechanism of action of PRP/Colostrinin in AD has not been yet clarified. It is known that oxidative stress and overproduction of NO may enhance neurodegenerative processes. PRP regulates the secretion of cytokines, inhibits NO and O2- release in cell cultures. Since the results on isolated cells or cell lines frequently do not reflect the events in vivo, the effect of PRP and its nonapeptide fragment (NP) on the level of NO2- in sera of mice untreated or intraperitoneally treated with LPS was studied. PRP and NP did not induce production of NO. However, when applicated 6 h after LPS, they inhibited the release of NO induced by LPS in about 30-50%. The results in vivo presented in this paper confirm the results obtained in cell cultures and indicate that the beneficial effects of PRP/Colostrinin observed in AD patients may be, among others, due to an inhibition of overproduction of NO.

Ovine colostrum nanopeptide affects amyloid beta aggregation

A colostral proline‐rich polypeptide complex (PRP) consisting of over 30 peptides shows beneficial effects in Alzheimers disease (AD) patients when administered in the form of sublinqual tablets called Colostrinin. The aim of the present studies was to investigate whether nanopeptide fragment of PRP (NP) – one of the PRP complex components can affect aggregation of amyloid β (Aβ1‐42). The effect of NP on Aβ aggregation was studied using Thioflavin T (ThT) binding, atomic force microscopy, and analyzing circular dichroism spectra. Results presented suggest that NP can directly interact with amyloid beta, inhibit its aggregation and disrupt existing aggregates acting as a β sheet breaker and reduce toxicity induced by aggregated forms of Aβ.

Immunomodulatory activity accompanying chicken egg yolk immunoglobulin Y

Abstract Immunity transfer from a mother to the newborn does not depend exclusively on immunoglobulins. Peptides, which are characterized by immunoregulatory properties that accompany IgG2, known as proline- rich polypeptide complex (PRP), have been discovered for the first time in ovine colostrum. In this report we present new data showing that some immunoregulatory peptides associated with the main immunoglobulin class, IgY, are also present in the avian immune system. Cytokine-inducing activity of particular fractions obtained from ovine colostrum, IgG+ (IgG2 containing PRP), IgG− (IgG2 free of PRP), and purified PRP, was compared with that of crude egg yolk IgY (IgY+), additionally purified egg yolk IgY (IgY−), and polypeptides accompanying IgY named Yolkin (Y), using an ex vivo model of whole human blood cells. It was shown that both IgG+ fraction and PRP, but not IgG−, stimulated the whole blood cells to release tumor necrosis factor-α and interleukin-1β cytokines. Similar experiments performed with hens egg IgY preparations showed that IgY+ and Y samples showed higher cytokine-inducing activity than samples additionally purified with the use of size exclusion chromatography (IgY−). The IgY+ at a dose of 100 μg was even more active than the positive lipopolysaccharide control. It was also found that Y is able to stimulate macrophage cell line J774.2 to release nitric oxide. The results obtained suggest that IgY, the main chicken immunoglobulin fraction, is accompanied by additional polypeptides and plays a role of a transporter of biologically active substances, which was observed in the case of colostral IgG.

Studies on the mechanism of action of a proline-rich polypeptide complex (PRP): Effect on the stage of cell differentiation

A proline-rich polypeptide complex (PRP) with immunoregulatory and procognitive activities shows beneficial effects in Alzheimers disease (AD). The mechanism of action of PRP in AD is not yet clarified. Here, we present results of the effect of PRP on Vitamin D3-induced phenotypic (CD11b and CD14) and functional (phagocytic) differentiation/maturation of monocytes/macrophages using the premonocytic HL-60 cell line as a model. This cell line can be induced to differentiate into monocyte/macrophage cells by incubation with Vitamin D3. However, when Vitamin D3 was applied together with PRP, a 30-40% inhibition of the expression of the differentiation markers and an over-60% inhibition of phagocytic ability were observed. When PRP was administered to the cells after treatment with Vitamin D3, no attenuation of the differentiation/maturation process of the HL-60 cells was observed. This indicates that PRP affects the early stages of differentiation/maturation of these cells. Our results, therefore, suggest that PRP, which affects the differentiation/maturation processes of cells of monocyte/macrophage lineage, may regulate in this way the inflammatory processes in which these cells participate.