Immunostimulating Substances from Human Casein

Abstract

Delipidated human casein was digested with trypsin and the enzymatic digest was fractionated on Sephadex G-50. The peptidic fractions were assayed for immunomodulating activity in two in vitro models. Fractions corresponding to molecular weights in the range of 2,000 +/- 600 were found to possess stimulating activity in these models.

Immunostimulating hexapeptide from human casein: amino acid sequence, synthesis and biological properties

“…In a previous publication [4] we reported that peptide fractions corresponding to relative molecular masses of 2000 600 and obtained through fractionation of an enzymatic digest of human casein exerted a stimulating activity in two tests of immunopharmacological activity in vitro, namely secretion of antibodies against sheep red blood cells (SRBC) by mouse spleen cells and phagocytosis of SRBC by mouse peritoneal macrophages. In the present paper, we describe further attempts at purifying the active component of these fractions, which led to the isolation and identification of the hexapeptide Val-Glu-Pro-Ile-Pro-Tyr.…”

“…HPLC, high-pressure liquid chromatography; SRBC, sheep red blood cells; Hepes, 4-(2-hydroxyethyl)-l -piperazine ethane sulfonic acid; MEM. Eagle's minimal essential medium; NaCI/ Pi.…”

“…Corp.). The digest was filtered on Sephadex (3-50 (Pharmacia) as previously described [4]. Further purification was monitored with various biological tests (see below) and was achieved according to the following procedure: (a) chromatography on a column (1 10 x 1.4 cm) of DEAE-Sephadex A-25 (Pharmacia) eluted successively with 0.01 M Tris/HCl, pH 8 (276 ml), 0.01 M Tris/HCl, pH 3.5, 0.1 M NaCl (300 ml) and 0.1 M Tris/HCl, pH 3.5, 0.5 M NaCl (530 ml); (b) NaCl filtration on a UM 05 Diaflo membrane (Amicon) followed by filtration on a column (120 x 1.2 cm) of Sephadex G-I 5 (Pharmacia) with 0.1 M acetic acid as eluent; (c) stepwise chromatography on a column (14 x 1.4 cm) of Dowex 50x4 (Bio-Rad) eluted successively with 0.05 M HCl (100 ml), H 2 0 (100 ml) and 2 M NH,OH (120 ml); (d) HPLC (Waters chromatograph, model ALC/ GPC 204) on a column (30 x 0.78 cm) of pBondapak C I 8 (Waters associates) eluted at a flow rate of 1 ml/min with 0.1% phosphoric acid (buffer A) during 10 min, then with a linear gradient of 70% acetonitrile (Baker Chemicals) in buffer A (buffer B) to reach 50% of each buffer within 100 min followed by a linear gradient from 50% to 100% buffer B within 30 min.…”

“…Maternal milk is usually man's first food: we previously observed that enzymic fragments obtained from human casein exhibit iminunostimulating properties [4]. The present paper describes the purification, the sequence, the synthesis and some biological properties of a hexapeptide from human casein.…”

Immunostimulating Agents: What Next?

“…Casein has been described as a milk protein with diverse biologic consequences [93] : casein fragments exert a rather wide range of immunopharmacological, hematological and neuropharmacological effects. Several years ago, our team observed that enzymic fragments obtained from human casein (maternal milk being frequently and advantageougly man's first food) exhibited immunostimulating activities, such as stimulation of phagocytosis by macrophages and enhancement of resistance of mice to a bacterial infection [94]. The purification, sequence, synthesis and further biological activities of an immunostimulating hexapeptide (Val-Glu-ProIle-Pro-Tyr) from human p-casein were described [95].…”