Biological role of a neurotrophic factor fragment from pigment epithelium: structure-functional homology with a differentiation factor for the HL-60 cell line

It was shown that the full-size neurotrophic factor from pigment epithelium (PEDF) induces the cell differentiation of the human promyelocyte leukemia cell line HL-60. A structural analysis of PEDF revealed in its C-terminal region a six-membered peptide fragment PEDF-(352-357) (PEDF-6) whose sequence is highly homologous to the 41-46 fragment of the active site of the human leukocyte differentiation factor HLDF (HLDF-6). The biological effect of PEDF and synthetic peptides PEDF-6 and HLDF-6 on the HL-60 cells and the early gastrula ectoderm of Xenopus laevis embryos was studied. On the basis of the structural and functional homologies of HLDF, PEDF, and their homologous peptides and the computer models of the spatial structures of the full-size PEDF and the PEDF with the C-terminal fragment split off tby the cleavage of the Leu380-Thr381 bond in the serpin loop, a hypothesis on the functional role of the serpin loop in PEDF was put forward.     

Different Mechanisms of Protective and Differentiative Activities of Homological Peptides TGENHR and TQVEHR

Previously we identified a six-membered fragment 354TQVEHR359 of the C-terminal part of the PEDF (Pigment Epithelium-Derived Factor) differentiation factor molecule that shares homology with fragment 41TGENHR46 of the HLDF (Human Leukemia Differentiation Factor) differentiation factor molecule, which is responsible for its differentiation activity. HLDF has been isolated from the culture medium of human promyelocytic leukemia cell line HL-60. Hexapeptides HLDF-6 (TGENHR) and PEDF-6 (TQVEHR) corresponding to these HLDF and PEDF molecule fragments, which were previously shown to induce cell differentiation (Kostanyan et al. (2000) Russian Journal of Bioorganic Chemistry, 26, 505-511), also have neuroprotective properties. Both peptides prevent degeneration of Purkinje cells of rat cerebellar vermis upon chemical hypoxia induced by sodium azide in vivo; this effect is also observed on a behavioral level. Peptide HLDF-6 but not PEDF-6 promotes survival of HL-60 cells upon chemical hypoxia. Peptides HLDF-6 and PEDF-6 affect different second messenger biosynthesis systems in HL-60 cells. HLDF-6 diminishes cyclic AMP level in those cells due to adenylate cyclase inhibition, while PEDF-6 inhibits phosphatidylinositol-specific phospholipase C stimulated by aluminum tetrafluoride anions.     

Effect of Homologous Peptides of Differentiation Factors HLDF and PEDF on Preimplantation Development of Mice in vitro

We studied the effect of synthetic peptides PEDF-6 and HLDF-6 on preimplantation development of mouse embryos in vitro. PEDF-6 peptide corresponds to fragment 351–356 and of pigment epithelium-derived differentiation factor (PEDF), while HLDF-6 peptide corresponds to fragment 84–89 of differentiation factor HLDF isolated from HL-60 cell line. Despite high homology, these peptides had different effects on the early development. PEDF-6 had no effect on the cleavage of 2–4-cell embryos but decelerated blastocyst formation from such embryos and disturbed their structure. In the presence of HLDF-6 the blastomeres divided more actively as compared to the control and a higher number of embryos developed to the blastocyst stage. The effects of both peptides were stage-specific: the affect the embryos at early cleavage stages and, apparently, determine their further development at that moment although do not directly affect formation of the blastocysts.     

Effect of homologous peptides of differentiation factors HLDF and PEDF on preimplantation development of mice in vitro

We studied the effect of synthetic peptides PEDF-6 and HLDF-6 on preimplantation development of mouse embryos in vitro. PEDF-6 peptide corresponds to fragment 351-356 and of pigment epithelium-derived differentiation factor (PEDF), while HLDF-6 peptide corresponds to fragment 84-89 of differentiation factor HLDF isolated from HL-60 cell line. Despite high homology, these peptides had different effects on the early development. PEDF-6 had no effect on the cleavage of 2-4-cell embryos but decelerated blastocyst formation from such embryos and disturbed their structure. In the presence of HLDF-6 the blastomeres divided more actively as compared to the control and a higher number of embryos developed to the blastocyst stage. The effects of both peptides were stage-specific: the affect the embryos at early cleavage stages and, apparently, determine their further development at that moment although do not directly affect formation of the blastocysts.     

A biologically active fragment of the differentiation factor of the HL-60 line cells: Identification and properties

Six-membered peptide fragment TGENHR (HLDF-6) was identified in the HL-60 cell culture of human promyelocyte leukemia treated with retinoic acid when studying the differentiation factor HLDF of this cell line. HLDF-6 retains the ability of the full-size factor to induce the differentiation and arrest the proliferation of the starting HL-60 cells. It was shown that the synthetic peptide HLDF-6 has no specific receptors on the surface of the HL-60 cells but can affect the binding of interleukin IL-1β, a cytokine involved in proliferation, to the cell surface. It was found on a model of transplantable NSO myeloma that HLDF-6 has an antitumor activity.     

Biologically active fragment of the differentiation factor from HL-60 cell line. Identification and properties

Six-membered peptide fragment TGENHR (HLDF-6) was identified in the HL-60 cell culture of human promyelocyte leukemia treated with retinoic acid when studying the differentiation factor HLDF of this cell line. HLDF-6 retains the ability of the full-size factor to induce the differentiation and arrest the proliferation of the starting HL-60 cells. It was shown that the synthetic peptide HLDF-6 has no specific receptors on the surface of the HL-60 cells but can affect the binding of interleukin IL-1 beta, a cytokine involved in proliferation, to the cell surface. It was found on a model of transplantable NSO myeloma that HLDF-6 has an antitumor activity.     

The Precursor of Differentiation Factor HLDF and Ribosomal Protein RPS21 Have a Common N-Terminal Sequence

The mature differentiation factor HLDF, isolated from cultural medium, comprises 54 aa, whereas the open reading frame of mRNA encodes a 97-aa protein. We presumed that the protein translation begins from the first ATG codon, whose environment mostly meets the requirements for the initiation point. Two more ATG triplets are localized in positions 48–50 and 100–102 (numbering according to the structure of S21), i.e., in the area preceding the cDNA fragment that encodes the N-terminal fragment of the mature protein. The mRNAs of HLDF and S21 ribosomal protein have previously been shown to be highly homologous, and, therefore, their differences appear to be derived from two point deletions in the cDNA of the HLDF-encoding sequence (a G residue in position 112 and a C residue in position 224). As a result, the mature differentiation factor and RPS21 may be the products of translation from different open reading frames, the differentiation factor may be synthesized in the cell as a precursor, and its N-terminal sequence may be identical to that of RPS21. To test this hypothesis, we prepared recombinant RPS21 and the polyclonal antibodies to HLDF, full-size RPS21, and the C-terminal RPS21 peptide. Immunochemical staining by specially produced antibodies of native HL-60 cells and the same cells brought into apoptosis or differentiation confirmed that the precursor of the differentiation factor and the ribosomal S21 protein have a common N-terminal sequence and different cellular localizations. Neither an intron-containing gene nor a pseudogene with the nucleotide sequence corresponding to the HLDF cDNA was detected in the human genome or in the HL-60 cell line genome. On the basis of these facts, we propose a hypothesis of the molecular mechanism of the HLDF mRNA biosynthesis by means of posttranslational modifications of pre-mRNA of RPS21.     

The nuclease activity of the endogenous differentiation factor of the HL-60 cell line

A structural homology between the endogenous differentiation factor of the HL-60 cell line of promyelocyte leukemia (HLDF) and several DNA/RNA-binding and DNA/RNA-hydrolyzing proteins was revealed, and expression of thehldf gene in prokaryotic systems was studied. On the basis of these experiments, the amino acid sequence of an 8-membered fragment of HLDF with potential nuclease activity was identified. The synthetic octapeptide RRWHRLKE was shown to be capable of the cleavage of RNA, linear DNA from phage λ, and all forms of plasmid DNA. We established that treatment of the HL-60 cell culture with this peptide (10⁻⁶ M) results in an increase in the number of apoptotic cells and suggested that HLDF is involved in processes of apoptosis.     

The endogenous differentiation factor of the HL-60 cells shows a nuclease activity

A structural homology between the endogenous differentiation factor of the HL-60 cell line of promyelocyte leukemia (HLDF) and several DNA/RNA-binding and DNA/RNA-hydrolyzing proteins was revealed, and expression of the hldf gene in prokaryotic systems was studied. On the basis of these experiments, the amino acid sequence of an 8-membered fragment of HLDF with potential nuclease activity was identified. The synthetic octapeptide RRWHRLKE was shown to be capable of the cleavage of RNA, linear DNA from phage lambda, and all forms of plasmid DNA. We established that treatment of the HL-60 cell culture with this peptide (10(-6) M) results in an increase in the number of apoptotic cells and suggested that HLDF is involved in processes of apoptosis.     

Precursors of HLDF differentiation factor and ribosomal protein RPS21 have a common N-terminal sequence

The mature differentiation factor HLDF, isolated from culture fluid, comprises 54 aa, whereas the open reading frame of mRNA encodes a 97-aa protein. We presumed that the protein translation begins from the first ATG codon, whose environment mostly meets the requirements for the initiation point. Two more ATG triplets are localized in positions 48-50 and 100-102, i.e., in the area preceding the cDNA fragment that encodes the N-terminal fragment of the mature protein. The mRNAs of HLDF and the S21 ribosomal protein have previously been shown to be highly homologous, and, therefore, their differences appear to be derived from two point deletions in the cDNA of the HLDF-encoding sequence (a G residue in position 112 and a C residue in position 224). As a result, the mature differentiation factor and RPS21 may be the products of translation from different open reading frames, the differentiation factor may be synthesized in the cell as a precursor, and its N-terminal sequence may be identical to that of RPS21. To test this hypothesis, we prepared recombinant RPS21 and the polyclonal antibodies to HLDF, full-size RPS21, and the C-terminal RPS21 peptide. Immunochemical staining by specially produced antibodies of native HL-60 cells and the same cells brought into apoptosis or differentiation confirmed that the precursor of the differentiation factor and the ribosomal S21 protein have a common N-terminal sequence and different cellular localizations. Neither an intron-containing gene nor a pseudogene with the nucleotide sequence corresponding to the HLDF cDNA was detected in the human genome or in the HL-60 cell line genome. On the basis of these facts, we propose a hypothesis of the molecular mechanism of the HLDF mRNA biosynthesis by means of posttranslational modifications of pre-mRNA of RPS21. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2004, vol. 30, no. 2; see also http://www.maik.ru.     

Proteolytic Hydrolysis of the Antitumor Peptide HLDF-6-AA in Blood Plasma

Russian Journal of Bioorganic Chemistry - HLDF-6 hexapeptide, which corresponds to the 41TGENHR46 fragment of human leukemia differentiation factor (HLDF), shows a wide range of neuroprotective,...     

Neuroprotective effect of the hexapeptide HLDF-6 on rat hippocampal neurons on the in vivo and in vitro models of alzheimer’s disease

The neuroprotective effect of Thr-Gly-Glu-Asn-His-Arg hexapeptide (HLDF-6), a biologically active fragment of the differentiation factor of human leukemia cells (HLDF), was demonstrated on models of Alzheimer’s disease in vivo and in vitro. The syndromes of this pathology were induced in male rats by injection of beta-amyloid peptide (25–35) and ibotenic acid into the hippocampus. HLDF-6 prevented loss of long-term memory and decrease in the exploratory behavior of these animals and significantly decreased the number of pyknotic neurons in the CA1 area of the hippocampus. This peptide also exerts a protective effect in vitro on the primary cultures of the rat hippocampal and cerebellar neurons under conditions of the beta-amyloid toxicity. An increase in the dihydrotestosterone (DHT) content was demonstrated in the blood plasma of rats with the syndrome of Alzheimer’s disease and in the medium of the culture of hippocampal neurons in the presence of the Aβ(25–35) peptide. HLDF-6 inhibited this increase in both cases. A probable mechanism of the neuroprotective effect of HLDF-6 was suggested as being connected to its possible effect on both the biosynthesis and the metabolism of sex steroid hormones.     

Investigation of the Hydrolytic Stability of the HLDF-6-AA Antitumor Peptide by the Method of Accelerated Aging

Russian Journal of Bioorganic Chemistry - The HLDF-6 hexapeptide corresponded to the 41–46 (TGENHR) fragment of the Human Leukemia Differentiation Factor (HLDF) and exhibited a wide spectrum...     

PEDF and the serpins: phylogeny, sequence conservation, and functional domains

Pigment epithelium derived factor (PEDF) is non-inhibitory serpin with neurotrophic and antiangiogenic functions. In this study, we have assembled PEDF sequences for 9 additional species by data base mining and performed cross-species alignment for 14 PEDF sequences to identify conserved structural domains. We found evolutionary conservation of a leader sequence, a single C-terminal glycosylation site, collagen-binding residues, and four specific conserved PEDF peptides. The C-terminus, 384--415 and an N-terminal region 78--95, show close homology with many other serpins, and there is strong conservation of 39 of 51 consensus key residues involved in serpin structure and function. Two peptide regions, 40--67 and 277--301, are unique to PEDF but conserved in all species. Conserved residues at the N-terminus, helix d (hD), and helix A (hA) of PEDF form a structure similar to the heparin-binding groove of other serpins. We identified a motif in PEDF that is homologous to the nuclear localization signals of other proteins. A bitopographical localization of PEDF was confirmed by immunocytochemistry and Western blots. Our results suggest that secretion is required for PEDF's activity, that PEDF can migrate to the nucleus, and that PEDF has structural and functional features more common with inhibitory serpins.     

HLDF-6 peptide affects behavioral reactions and organism functions dependent on androgen hormones in normal and castrated male mice

The hexapeptide Thr-Gly-Glu-Asn-His-Arg (HLDF-6), which was first identified as an active fragment of the human leukemia differentiation factor (HLDF) molecule, displays differentiation-inducing, neuroprotective and anti-drug abuse activities. Most of its in vivo effects were revealed only on male animals. We have studied HLDF-6 effects on a variety of organism functions and behavioral reactions, which are known to be dependent on androgen steroid hormones, both on castrated and normal (sham-operated) animals. Male NMRI mice were castrated or sham-operated at the age of 55 days (after puberty). After that, HLDF-6 peptide was injected daily during 3 weeks, followed by behavioral, morphological and biochemical testing. HLDF-6 increased testosterone level (1.5- to 2-fold) both in sham-operated and castrated animals. Sexual activity and pain sensitivity, which are strongly reduced in castrates, were completely or partially recovered by HLDF-6. At the same time, the peptide caused some effects similar to castration in sham-operated animals: aggression and locomotor activity were decreased; oral grooming was prolonged. Morphological studies of accessory sex glands showed that HLDF-6 partially normalizes the morphology and functional activity of seminal vesicles in castrates, but it does not prevent castration-induced apoptosis of prostate epithelial cells. Based on these observations, we can assume that HLDF-6 peptide displays at least two effects on androgen hormones metabolism in males: it stimulates testosterone biosynthesis by both testes and adrenals and simultaneously inhibits its conversion to dihydrotestosterone (DHT), most probably by diminution of 5alpha-reductase isoform 1 mRNA expression.     

Neuroprotective effect of the hexapeptide HLDF-6 on neurons of rat hippocampus on the model of Alzheimer's disease in vivo and in vitro

The neuroprotective effect of Thr-Gly-Glu-Asn-His-Arg hexapeptide (HLDF-6), a biologically active fragment of the differentiation factor of human leukemia cells (HLDF), was demonstrated on models of Alzheimer's disease in vivo and in vitro. The syndromes of this pathology were induced in male rats by administration of the peptide corresponding to the 25-35 sequence of beta-amyloid peptide (25-35) and ibotenic acid into the hippocampus. HLDF-6 prevented loss of long-term memory and decrease in the orientation-investigation activity of these animals and significantly decreased the number of pyknotic neurons in the CA1 area of the hippocampus. This peptide also exerts a protective effect in vitro on the primary cultures of neurons of the hippocampus and cerebellum of rats under conditions of the beta-amyloid toxicity. An increase in the dihydrotestosterone (DHT) content was demonstrated in the blood plasma of rats with the syndrome of Alzheimer's disease and in the medium of the culture of hippocampus neurons in the presence of the Abeta(25-35) peptide. HLDF-6 inhibited this increase in both cases. A probable mechanism of the neuroprotective effect of HLDF-6 was suggested as being connected to its possible effect on both the biosynthesis and the metabolism of sex steroid hormones.     

An Artificial Protein with the Biological Activity of the Differentiation Factor for the HL-60 Cell Line of Human Promyelocyte Leukemia

The ABB-df artificial protein was prepared by inserting the TGENHR biologically active peptide corresponding to the 41–46 sequence of the differentiation factor for the HL-60 cell line of the human promyelocyte leukemia into the N-terminus of the polypeptide chain of albebetin, an artificial protein with the preset structure. The ABB-df protein was found to induce the differentiation of HL-60 cells and to inhibit their proliferation; its efficiency was almost the same as that of the starting peptide. According to CD spectroscopy, the inclusion of the peptide fragment into albebetin exerts virtually no effect on the regular secondary structure of albebetin.     

Mapping the type I collagen-binding site on pigment epithelium-derived factor. Implications for its antiangiogenic activity

Pigment epithelium-derived factor (PEDF), a neurotrophic and antiangiogenic serpin, is identified in tissues rich in collagen, e.g. cornea, vitreous, bone, and cartilage. We show that recombinant human PEDF formed complexes with collagens from the bovine cornea and vitreous. We have examined the direct binding of PEDF to collagen I and found that interactions were ionic in nature and occurred when PEDF and collagen I were both in solution, when either one was immobilized, or even when collagen I was denatured under reducing conditions. (125)I-PEDF bound to immobilized collagen I in a saturable fashion (K(D) = 123 nm). Compared with neurotrophic PEDF-derived peptides, ovalbumin and angiogenic inhibitors, only full-length PEDF competed efficiently with (125)I-PEDF for the binding to immobilized collagen I (EC(50) = 3 microg/ml). The collagen-binding region was analyzed using controlled proteolysis and chemically modified PEDF. Cleavage of the serpin exposed loop did not prevent binding to collagen I. Conjugation of lysines with fluorescein increased the collagen binding affinity. However, treatment of PEDF with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide abolished it, implicating the PEDF aspartic and/or glutamic acid residues in its interaction with collagen I. A negatively charged region on the surface of the PEDF molecule is rich in acidic residues (Glu(41), Glu(42), Glu(43), Asp(44), Asp(64), Asp(256), Asp(258), Glu(290), Glu(291), Glu(296), Asp(300), Glu(304)) available to interact directly with positively charged areas of collagen. This represents the first collagen-binding site described for a serpin, which in PEDF, is distinct from its heparin-binding region, neurotrophic active site, and its serpin exposed loop. The collagen-binding property of PEDF may play a role in surface localization and modulation of its antiangiogenic effects in the eye and bone.     

Identification and expression of haponin, a new protein from HL-60 cells

We found a new protein haponin (an HLDF-like protein) in promyelocyte HL-60 cells that is immunoreactive to polyclonal antibodies against HLDFβ. Determination of the partial primary structure of the protein allowed us to reveal an immunogenic peptide of haponin and, on the basis of the amino acid sequence of this peptide, the degenerate primers were synthesized, which enabled us to clone the full-size cDNA of haponin. The stable heterologous expression of this cDNA in E. coli cells (Rosetta? strain) was obtained. Preparations of natural and recombinant proteins exhibited antigenic cross-reactivity to polyclonal antibodies against this peptide.