Marina M. Philippova

Proteolytic degradation of hemoglobin in erythrocytes leads to biologically active peptides

A number of hemoglobin-derived homogeneous peptides were isolated from erythrocyte lysate. The amino acid sequences of nine peptides were determined. Seven out of nine peptides were relatively long, 30-32 membered peptides covering the N- or C-terminal sequences of globin chains. The remaining two were the pentapeptide neo-kyotorphin and its tetrapeptide derivative.

Family of hemorphins: co-relations between amino acid sequences and effects in cell cultures.

Hemorphins, i.e. endogenous fragments of beta-globin chain segment (32-41) LVVYPWTQRY(F) suppress the growth of transformed murine fibroblasts L929 cell culture, the effect is due to cytotoxicity and inhibition of cell proliferation. The contribution of cytotoxicity depends on the presence of Leu(32): VV-hemorphins, except VV-hemorphin-4, exhibit cytotoxicity significantly higher than respective LVV-hemorphins. Decrease of cell number induced by hemorphins depend on the extent of N- and C-terminal degradation of hemorphins: VV-hemorphins in most cases are more active than LVV-, V-hemorphins, and hemorphins. In the group of VV-hemorphins the activity of VV-hemorphin-5 (valorphin) is significantly higher than of VV-hemorphin-7, VV-hemorphin-6, and VV-hemorphin-4, meaning that the presence of C-terminal Gln is important for suppressing of cell number. The amino acid sequence VVYPWTQ corresponding to valorphin was identified as important for manifestation of the both cytotoxic and antiproliferative effects.

LVV‐ and VV‐hemorphins: comparative levels in rat tissues

Screening of hemorphins in extracts of rat lung, brain, heart and spleen was carried out. The threshold for detection of hemorphins was 0.01 nmol for spleen and 0.05 nmol for other tissues. Both the content and the composition of hemorphins differed significantly in the tissues analyzed. Heart and lung extracts were rich in these peptides, the content of the most abundant components reaching 16–44 nmol/g of tissue. In contrast, spleen and brain contained much lower amounts of hemorphins, i.e. about 0.3–2.6 nmol/g of tissue. The most represented hemorphin in lung, heart and brain was VV‐hemorphin‐5, while the content of other members of the hemorphin family depended significantly on the tissue analyzed: lung extract was also rich in LVV‐hemorphin‐5, heart contained similar amounts of LVV‐hemorphin‐7 and LVV‐hemorphin‐5 and brain of LVV‐hemorphin‐6. In contrast, the hemorphin family in spleen was represented mainly by C‐terminally shortened VV‐hemorphins, i.e. VV‐hemorphin‐4 and VV‐hemorphin‐3. The levels of hemorphins in all cases were sufficient to activate the opioid receptors of the respective tissues.

Neokyotorphin and neokyotorphin (1–4): secretion by erythrocytes and regulation of tumor cell growth

Human erythrocytes release neokyotorphin, the 137–141 fragment of hemoglobin α‐chain into the supernatant of red blood cells primary culture. However, the neokyotorphin fragment 1–4 that is formed together with neokyotorphin inside the red blood cells and in various tissues is not found in the supernatant. Both neokyotorphin and its 1–4 fragment were shown to stimulate proliferation of L929 tumor cells.

Peptides from bovine brain: structure and biological role.

Fractionation of bovine brain extracts followed by automatic Edman sequencing of individual components resulted in identification of 107 endogenous peptides formed from functional proteins (haemoglobin, myelin basic protein, cytochromecoxidase, etc) or unknown precursors. Several of the newly identified brain peptides demonstrate different types of biological activity; some of the substances show considerable overlap with the known biologically active peptides. It is suggested that these peptides should participate in regulation of extracellular and intracellular biochemical processes. A concept of ‘tissue‒specific peptide pool’ is formulated describing a novel system of peptidergic regulation, complementary to the conventional hormonal and neuromodulatory systems. According to that description functional proteins provide their proteolytically derived fragments for maintaining the tissue homeostasis by modulating the availability of peptide receptors to respective ‘true’ ligands.

Peptides comprising the bulk of rat brain extracts: isolation, amino acid sequences and biological activity

Chromatographic separation of rat brain extracts followed by automatic Edman sequencing of the major individual components resulted in identification of 61 endogenous peptides derived from known functional proteins (hemoglobin, myelin basic protein, cytochrome‐c oxidase, etc.) or unknown precursors. The results are compared with the data obtained earlier for bovine brain. Although the sequences of bovine and rat hemoglobin contain about 20% of amino acid substitutions, the families of structurally related peptides are very similar in both extracts. Several other proteins also give rise to identical or closely related peptide fragments in the two mammalian species. The outlined similarity extends almost exclusively to the most abundant peptides present in the extracts. The minor components show less overlap. Four hemoglobin‐derived peptides isolated from rat brain were shown to be biologically active in tumor cells. Eleven are identical to bioactive peptides from other species. Ten structurally overlap with bioactive peptides from other sources. The data obtained show similar biosynthetic pathways of pool components in different species, the resultant peptides being aimed at fulfilling related functions. Copyright

Endogenous fragment of hemoglobin, neokyotorphin, as cell growth factor.

It is shown that neokyotorphin (the alpha-globin fragment 137-141) stimulates proliferation of normal cells (murine embryonic fibroblasts, red bone marrow and spleen cells) and tumor cells (murine melanoma and transformed fibroblasts L929) in the absence or in the presence of fetal bovine serum. In contrast to serum deprivation conditions, the ability to potentiate L929 cell growth in the presence of fetal serum is strongly cell density dependent. The peptide also enhances the viability of L929 cells, murine embryonic fibroblasts and of the primary cultures of murine red bone marrow cells and splenocytes under serum-deprivation conditions for at least 72 h. The results of flow cytometry analysis suggest that the effect of neokyotorphin on survival of L929 cells in serum-free culture medium is due to maintenance of cell proliferation in the absence of growth factors. Along with cell cycle progression the peptide induces reversible reduction of L929 cell size.

Proliferative Activity Of Neokyotorphinrelated Hemoglobin Fragments In Cell Cultures

alpha-Hemoglobin fragments alpha-(133-141), alpha-(134-141), alpha-(135-141), alpha-(137-141), alpha-(134-140), alpha-(133-138), alpha-(134-140) and alpha-(137-138) stimulate L929 tumor cell proliferation, alpha-(134-141) being the most active. alpha-(134-141) stimulates proliferation of M3 melanoma cells, murine embryonic fibroblasts, primary cultures of red bone marrow and spleen cells. In L929 cells the effect of alpha-(134-141) is cell density independent; in M3 cells alpha-(137-141) and alpha-(134-141) are most active at density 10,000 cells/well (96 well plate) independently on FBS content.

Alteration of Intraerythrocyte Proteolytic Degradation of Hemoglobin During Hodgkin's Disease

The erythrocyte lysate samples obtained from 10 healthy donors (aged of 23 +/- 12 years) and 16 patients with Hodgkins disease (aged of 39 +/- 25 years) with the following histological types: 12 mixed cellularity and 4 nodular sclerosis, were studied. Patients with Hodgkins disease (HD) were randomly selected before, during or after the completion of combined chemotherapy. A comparative analysis of peptide components of erythrocyte lysate samples of HD patients and healthy donors was carried out. The amino acid sequences of 4 peptides, corresponding to fragments 1-33, 1-32, 1-31 and 1-30 of human hemoglobin (Hb) alpha-chain were determined. Increase of the content of two fragments corresponding to 1-31 and 1-32 amino acid residues of alpha-globin were detected for HD patients. The link between the normal process of proteolytic degradation and those occurring during HD is proposed. The possibility of using the identified alterations recorded during HD diagnosis is discussed.

Fragments of functional proteins in a primary culture of human erythrocytes

According to the previously reported data, the superntant of the primary culture of human erythrocytes contains 33 hemoglobin fragments. An analysis of the supernatant of a 20% (v/v) suspension of human erythrocytes allowed us to identify additionally four peptides whose precursors are cytoplasmic β-actin (two fragments), fructose diphosphate aldolase B, and an unknown protein, and amino acids tyrosine and tryptophan. The composition and the content of the components of the supernatant did not depend on the age and blood group of donors. The dynamics of accumulation in the supernatant (20–80 min of incubation) of 14 hemoglobin fragments with the most reliably reproducible contents was obtained. The content of six peptides increased more than twofold between 20 and 40 min of incubation; the maximum increase in concentration was observed between 40 and 80 min (140%). The level of peptides that had the maximum concentration at the end of incubation was about 1000 pmol/ml of sedimented erythrocytes. The biological effects of the peptides identified in the supernatant of erythrocytes involve the stimulation of proliferation and hemopoiesis, suppression of proliferation, a bactericide effect, etc. These effects indicate the physiological importance of the peptide release by erythrocytes.