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Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry Russian Academy of Sciences, Ul. Miklukho-Maklaya 16/10, 117871 Moscow, Russia
Hemoglobin as a Source of Endogenous Bioactive Peptides: The Concept of Tissue-Speciﬁc Peptide Pool
Abstract: Scattered literature data on biologically active hemoglobin-derived peptides are collected in the form of tables. Respective structure–functional correlations are analyzed and the general conclusion is reached that hemoglobin fragments must have a profound physiological function. Evidence is presented that generation of hemoglobin fragments starts inside the erythrocytes. At that stage a- and b-globin chains of hemoglobin predominantly give rise to relatively long peptides containing ca. 30 amino acid residues. The primary proteolysis is followed by the next degradation step coupled with excretion of newly formed shorter peptides form red blood cells. Both the primary and the secondary proteolysis products are subjected to further stepwise C- and N-terminal chain shortening, giving rise to families of closely related peptides that are actually found in animal tissue extracts. The possible sites of primary proteolysis are compared with the positions of the exposed secondary structure elements within the monomeric a- and b-globins as well as the tetrameric hemoglobin. Two tentative schemes are proposed for hemoglobin degradation, one of which starts at the globin loops exposed on the surface of the tetramer and the other, at monomeric globins where more sites are available for the action of proteases. The concept of a ‘‘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, hemoglobin is only a single example, although an important one, of a vast number of functional proteins providing their proteolytically derived 1997 John Wiley & Sons, Inc. Biopoly fragments for maintaining the tissue homeostasis. 43: 171–188, 1997 Keywords: hemoglobin-derived peptides; hemoglobin proteolysis in vivo; peptide excretion from erythrocytes; tissue homeostasis; tissue speciﬁc peptide pool
As early as 1971, Schally et al. in their pursuit of novel hypothalamic factors discovered in pig hypothalamus a decapeptide with growth hormone releasing activity.1 However, the interest in that pep-
tide faded as soon as it turned out to be a 1–10 fragment of the b-chain of hemoglobin. Moreover, other 4–16 membered peptides isolated later from the same source were also considered artefacts solely because they reproduced the amino acid sequences of a- or b-globin chains.2,3 The apparent
Correspondence to: Vadim T. Ivanov 1997 John Wiley & Sons, Inc.
The search for opioidlike hemoglobin fragments (hemorphin-containing peptides) obtained by in vitro proteolysis was continued in publications of Piot et al. lung. which therefore could originate from another protein. In 1986 Brantl et al. In part that was due to the short length of the abovementioned peptides. although their opioid properties have not been studied.8 Moreover.and d-opiate ligands to their receptors. Hemoglobin Fragments Isolated by Schally et al.16.. Systematic study of peptide composition of various tissue extracts attempted several years ago in our laboratory resulted in an avalanche of new peptide structures. i. In other words.39 Although the abovementioned myelopeptides induce naloxon-supressed analgesia in vivo. reviewed in this issue of Peptide Science.36 The list of endogenous peptides with hemoglobin amino acid sequences was extended by a group of coronaro-constrictory peptides from bovine hypothalamus (Table V). with opioid-like activity. We believe the minimal hemoglobin-derived unit displaying genuine opioid activity is the tetrapeptide Tyr-Pro-Trp-Thr. Neokyotorphin was shown to terminate the hibernation state in ground squirrels and to enhance the . many of these peptides originate from a. STRUCTURE AND BIOLOGICAL ACTIVITY OF HEMOGLOBIN-DERIVED PEPTIDES FROM TISSUE EXTRACTS Data on peptides mentioned in the introduction are summarized in Tables I–III.30 As seen from Tables VI–VIII. Only in 1990s did it become clear that hemoglobin serves in vivo as a powerful source of bioactive peptides that must have a profound role in homeostasis. peptides secreted by pig red bone marrow cell primary culture contains b-chain (32–37) peptide and a-chain (33– 38) peptide fragments.e. not all biological properties of peptides from the ‘‘strategic’’ b-chain (32–41) zone should be ascribed opioid receptor binding.40 Therefore one should also not expect any signiﬁcant opioid activity from the above-mentioned coronaro-constrictory b-chain (33–37) peptide shown in Table V. (1971–1980) from Pig Hypothalamus Fragment a a b b b b Sequence FLGFPTTKTYFPHFNL2 FLGFPTTKTYFPHF2 VHLSAEEKEA1 GKVN3 LVVYPWTQRF3 VVYPWTQRF3 Activity — Corticotropin release in vitro2 Growth hormone release in vivo and in vitro1 Prolactin release in vitro3 — — 33–48 33–46 1–10 16–19 32–41 33–41 although never explicitly formulated reason for such conclusion was the belief that since the function of hemoglobin as an oxygen carrier is ﬁrmly established.7 – 10 Since these experiments were done in vitro. the a-chain (33–38) peptide only weakly inhibited binding of m... Five of them. The family of myelopeptides. including that of Nyberg et al. called hemorphins. The results reviewed in this issue add another dimension to the spectrum of bioactivities of hemoglobin peptide fragments. i. and the b-chain (32–37) peptide was completely inactive in that test (IC50 ú 10 04 M).172 Table I Ivanov et al. also were not interpreted as an indication of the second function of hemoglobin. it remained uncertain whether similar processes occur in the living organism. positioned within the b-chain (32–41) segment. apparently belong to the hemorphin group.38 Both these peptides were studied in a variety of immunological test systems. and describes new results related to the mechanism of their biosynthesis. found that proteolytic treatment of hemoglobin gives rise to a series of peptides.e. Neokyotorphin and its fragment (1–4) were found in the brain of ground squirrels as well as in the brain.6 That pioneering work was followed by a series of communications dealing with proteolytic degradation of hemoglobin as well as with bioactive products resulting from such treatment.or b-globin chains. as described in his contribution to this issue. The present paper reviews the available data on the structure and properties of these peptides. one should not expect other signiﬁcant functions from the same protein. The subsequent discovery in bovine brain in 1979 and in 1982 of kyotorphin 4 and neokyotorphin 5 reproducing correspondingly the 140–141 and 137–141 C-terminal sequences of a-globin. bchain (35–38) fragment or hemorphin-4. hemorphincontaining peptides were found in vivo in several laboratories (Table IV). and heart of rat (Tables II and VI).
the huge amount of hemoglobin circulating inside the erythrocytes suggest itself as the most probable precursor of peptides listed in Tables I. the shortened by one C-terminal amino acid fragment inhibited the Ca 2/ current 42 and showed pronounced cytotoxic effects in the above-mentioned transformed cell lines. and IV–VIII.5% of the hemoglobin content. With the exception of neokyotorphin and its des-C-terminus derivative found in very minor amounts. the longest a-chain (1–33) peptide is by far the most abundant fragment present inside the erythrocytes. The overall amount of these peptides comprised ca.13 In contrast to neokyotorphin. giving rise to peptides shown in Table X.and b-globins.41 It is worth mentioning that one of these hemoregulatory peptides. it was not cytotoxic in human erythroid leukemia (K562) and murine-transformed ﬁbroblast (L929) cell lines. 0. The supernatant of erythrocytes incubated in the phosphate buffer (pH . a-globin (1–16). (Table IX). II..43 – 45 In summary. ERYTHROCYTES AS A PRIMARY SOURCE OF HEMOGLOBIN-DERIVED PEPTIDES The abundance of hemoglobin fragments in various tissues raises a question of the mechanism of their formation and delivery. In turn. spans three quarters of the sequence of the peptide a-globin (1–21) from Table V. In addition to the earlier published sequences. A number of hemoglobin-derived peptides were isolated from brain extracts by Sleemon et al. with sequences spanning the b-globin (32–41) sequence] were amply represented in the bovine brain extracts (Table VII).Hemoglobin as a Source of Peptides Table II Neokyotorphin Family of Hemoglobin Fragments Peptide YR Kyotorphin Fragment a 140–141 173 Source Bovine brain4 Rat spinal cord11 Rat brain11 Bovine brain5 Hibernating ground squirrel12 Rat brain13 Rat lung13 Rat heart13 Human lung carcinoma14 Human erythrocytes15 Hibernating ground squirrel16 Rat brain13 Rat lung13 Rat heart13 Human erythrocytes15 Hibernating ground squirrel16 Activity Analgesic4 TSKYR Neokyotorphin a 137–141 Analgesic5 Antihibernatic12 Ca2//K/ current regulation12 TSKY a 137–140 Ca2//K/ current regulation16 Cytotoxicity13 SKYR a 138–141 — inward potential dependent Ca 2/ current in cardiac myocytes of rat 42 .46 However. Several of the peptides from bovine red bone marrow were able to restore the hemopoietic function of mice subjected to radiation or treated with 5ﬂuorouracil (Table VIII). the latter also shows the relative concentrations of isolated peptides.30. The reason for the above-mentioned discrepancy was found in our recent study of peptides released by erythrocyte primary culture. it was shown that some of these peptides can serve as markers of such pathologies as Alzheimer’s disease and brain ischemia. In principle. the globin genes can be expressed not only in red blood cells and therefore respective peptides might form directly within any cell. Although the structural approach applied in that work did not allow the determination of the complete amino acid sequences.13 Peptides belonging to the hemorphin family [i. hemoglobin fragments isolated from various sources cover almost the entire sequences of a. Of them. The current state of the above-mentioned relationship is illustrated in Figure 1. the intraerythrocytic peptides have notably longer sequences than most of the above-discussed endogenous hemoglobin fragments.e. a considerable part of these sequences display distinct bioactivity. Chromatographic analysis of the peptide content in human erythrocytes (Figures 2A and 3A) demonstrated that intensive proteolytic degradation of hemoglobin takes place under normal conditions.
show a very considerable overlap with respective peptides in Tables I. A striking feature of the obtained result is the fact that not a single peptide found inside erythrocytes was present in the supernatant. b-chain (1–6). Thirteen peptides were identiﬁed as hemoglobin fragments. As a result. Four of the peptides present in the supernatant occupy the same positions in the a. The overall content of peptides in the supernatant after 4. 7.174 Ivanov et al. peptide content of both. these peptides.. b-chain (34–37) peptide— coronaro-constrictory 28 and/or immunomodulatory 38 activities. b-(2–9) (Table VIII) and b-(133–146) (Table V). (Table XI) the remaining two (NDKVQPLE and KEATQE. not shown in Fig. 15 amino acid sequences of isolated peptides were established. as well as most of the other peptides from Table XI [with the exception of a-chain (84–88) and b-chain (87–91) fragments]. a-chain (1–17). the b-chain (34– 39) peptide is expected to display opioid properties (Table IV). pos- . sex. a-chain (1–12). a-chain (1–11). Accordingly. IV. and VIII. or age of the donor. the erythrolysate and the supernatant did not depend upon the blood group. and b-chain (72–84) peptides—hemopoietic activity 41 (Table VIII). 3B and Table XI) are derived from other proteins. b-(2–11). As with the erythrocytes lysates.2) was fractionated on size-exclusion (Figure 2B) and reverse phase high performance liquid chromatography (RP-HPLC. Figure 3B) columns. From that observation we conclude that release of peptides from erythrocytes is accompanied by proteolytic degradation of initially formed long peptide sequences. 2 in order to allow correct alignment with sequences from other animal species. the chromatographic proﬁles obtained with the supernatant did not show any signiﬁcant individual differences. i. Biologically Active Hemoglobin Fragments Obtained by In Vitro Proteolysisa Fragment a 110–125 a 129–134 b 32–41 Table III Peptide Proteolitic Treatment Bovine hemoglobin treated with pepsin7 Bovine hemoglobin treated with pepsin8 Bovine hemoglobin treated with pepsin9 Activity Bradykinin potentiation7 Bradykinin potentiation8 Speciﬁc opiate receptor binding17 Inhibition of electrically induced contractions of GPI9 Inhibition of ACE18 Coronaro-constrictory19 Speciﬁc opiate receptor binding17 Inhibition of electrically induced contractions of GPI9 Bitter peptide20 Inhibition of ACE18 Naloxon-depending analgesia21 Speciﬁc opiate receptor binding22 Inhibition of electrically induced contractions of GPI6 Naloxon-depending analgesia21 Inhibition of electrically induced contractions of GPI6 Inhibition of ACE10 Inhibition of ACE10 Inhibition of ACE10 ASHLPSDFTPAVHASL LANVST LVVYPWTQRF VVYPWTQRF b 33–41 Bovine hemoglobin treated with pepsin9 YPWT Hemorphin-4 b 35–38 Bovine hemoglobin treated with pepsin/cathepsin6 YPWTQ Hemorphin-5 b 35–39 Bovine hemoglobin treated with pepsin/cathepsin6 Pig hemoglobin treated with trypsin10 Pig hemoglobin treated with trypsin10 Pig hemoglobin treated with trypsin10 GKKVLQ FQKVVA FQKVVAG b 64–69 b 130–135 b 130–136 a In Table III and the following tables the numbering of bovine b-globin chain starts with No. VII. b-chain (1–10). b-chain (1–11) peptide—growth hormone release activity 1 . As a result.5 h incubation was ca. The obtained fractions were rechromatographed and substances corresponding to main peaks were analyzed by Edman technique. 2–5% of the peptide content inside erythrocytes.e.and b-globin chains as peptides found earlier in bovine extracts: a-(1–12).
a VVYPWTQR32. hemor- Fragment a a a a b b b b b b b 1–21 110–124 111–121 112–126 32–41 32–38 33–40 33–38 33–37 130–146 133–146 Table VI Hemoglobin Fragments Isolated from Brain of Hibernating Yakut Ground Squirrels Citellus undulatus16 Sequence VLSPA TSKYR TSKY SKYR VHLSDGEKNAISTAWG VHLSDGEKNAISTA IVIVMA VVAGVANA Fragment a a a a b b b b These peptides display coronaro-constrictory properties. energy.a LVVYPWT28. and IX) displays considerable differences. At the same time the array of concrete peptides found in three thus far studied tissues (bovine bone marrow.) require further investigation. VIII. and human cerebellum.a FQKVVAGVANALAHRYH32 VVAGVANALAHRYH37 a There are all grounds to assume that release of short peptides from erythrocytes makes an important contribution to the overall peptide content of various tissues. Sequence VLSAADKGNVKAAWGKVGGHA37 ASHLPSDFTAPAVAS37 SHLPSDFTPAV32 HLPSDFTPAVHASLD32 LVVYPWTQRF19.Hemoglobin as a Source of Peptides Table IV Sequence LVVYPWTQRF Hemorphins and Related Peptide Fragments of b-Globin Position 32–41 Source Pig hypothalamus3 Bovine hypothalamus19 Bovine brain23 Human liquor24 Human gingival crevicular ﬂuid25 Human pituitary gland26 Activity Speciﬁc opiate receptor binding17 Inhibition of electrically induced contractions of GPI9 Coronaro-constrictory activity19 Inhibition of ACE18 175 LVVYPWTQR 32–40 LVVYPWTQ LVVYPWT 32–39 32–38 VVYPWTQRF 33–41 Bovine brain23 Bovine brain23 Bovine hypothalamus28 Bovine spinal cord29 Pig hypothalamus3 Bovine brain23 Mice peritoneal macrophages31 VVYPWTQR VVYPWTQ 33–40 33–39 Bovine hypothalamus32 Bovine hypothalamus33 Bovine brain23 VVYPWT YPWTQRF 33–38 35–41 Bovine hypothalamus28 Human plasma35 Speciﬁc opiate receptor binding26 Inhibition of electrically induced contractions of GPI26 Inhibition of ACE27 — Coronaro-constrictory activity28 Inhibition of enkephalin-degrading enzyme29 Convulsion in vivo30 Speciﬁc opiate receptor binding17 Inhibition of electrically induced contractions of GPI9 Inhibition of ACE18 Bitter peptide20 Coronaro-constrictory activity32 Speciﬁc opiate receptor binding33 Inhibition of electrically induced contractions of GPI33 Cytotoxicity34 Coronaro-constrictory activity28 Speciﬁc opiate receptor binding9 Inhibition of electrically induced contractions of GPI17 sibly by membrane-associated proteases. etc. The particular type of protease as well as the mechanism of transmembrane passage (direct transport or secretion involving vesicle formation. 1–5 137–141 137–140 138–141 1–16 1–14 110–115 133–140 .a VVYPW28. For instance. bovine brain. see Tables VII. Table V Hemoglobin Fragments Isolated from Bovine Hypothalamus by Galoyan et al. and carrier requirement.a VVYPWT28.
Accordingly.1–1.0 0.1–1.0 1.1–1.0 0.0 0.0 0.1 0. For that purpose we collected in Figures 4 and 5 all peptides from Tables I–XI and aligned them under the a.1–1.0 0.1–1.176 Ivanov et al. speciﬁc set of hemoglobin-derived peptide components.0–3.0 1. we assume that long peptides con- taining ca.0 1.0 0.0–3.0 0.0 0.1–1.1–1.0 0.1–1.0 0.1–1. In other words.1–1.0 0.1–1.1–1.1–1. 30 amino acid residues and found in tissue extracts come from the intraerythrocyte pool of the peptides where they are represented in high amounts (Table X).0 0. PROTEOLYTIC DEGRADATION OF HEMOGLOBIN With more that 150 established amino acid sequences of endogenous hemoglobin fragments available for comparison.0 0.0 0. also speak of tissue speciﬁcity. data although rather fragmentary on the content of hemoglobin-derived peptides in various samples.0 0.0 0.1–1.0 0.0 õ 0. . The observed level of peptides in the tissue extract is a sum of the contributions from the erythrocytes always present in blood vessels and from the rest of the tissue.1–1.0–3.1–1. The shorter peptides are genuine tissue speciﬁc peptides and are either absorbed from the blood stream or result from proteolytic degradation of the absorbed peptides.0 0.0 0.1–1.1–1.1–1.1–1. The available quantitative. it is tempting to analyze the pathways of hemoglobin proteolysis in more detail. For instance.1–1.1–1.1–1. in spite of arguments favoring the role of erythrocytes as a common primary source of hemoglobin fragments. the level of neokyotorphin in rat lung is 4–5 times higher than in erythrocytes.13 We believe that peptides released from the erythrocytes either accumulate in the tissue surrounding the blood vessel or are further degraded by tissue-speciﬁc proteases.0 0.0 0. Table VII Hemoglobin Fragments Isolated from Bovine Brain30 Content (nmol/g Tissue) 0.0 1.0 0.1–1.0 0.and b-globin sequences of respective species.0–3.0 Sequence VLSAADKGNVKAAWGKVGGHAAEYGAEALERM VLSAADKGNVKAAWGKVGGHAAEYGAEALE VLSAADKGNVKAAWGKVGGHAAEY DKGNV LSHSL ASHLPSDFTPAVHASLDKFLANV ASHLPSDFTPAVHASLDK FLANVSTVL MLTAEEKAAVTAFWGKVKVDEVGGEALGRL MLTAEEKAAVTAFWGKVKVDEVGGEALG MLTAEEKAAVTAF MLTAEEKA MLT WGKVKVDEVGGEA WGKVKVDEVG EVGGEALG EVGGEAL GGE LVVYPWTQRF LVVYPWTQ LVVYPWT LVVYP VVYPWTQRF VVYPWTQ VVVLARNFGKFFTPVLQADFQKVVAGVAN-? VVVLARNFGKFFTPVLQ VVVL ARNFGKFFTPVLQ VLQ FQKVVAGVANALAHRYH a a a a a a a a b b b b b b b b b b b b b b b b b b b b b b Fragment 1–32 1–30 1–24 6–10 101–105 110–132 110–127 128–136 2–31 2–29 2–14 2–9 2–4 15–27 15–24 22–29 22–28 24–26 32–41 32–39 32–38 32–36 33–41 33–39 111–139? 111–127 111–114 115–127 125–127 130–146 phins were not found in bovine bone marrow extracts and are well represented in bovine brain ( ú1 nmol/g of tissue).1–1. each tissue has its own.
1 0.0 0.01 0.01–0.01–0.1 0.1 0.01–0.01–0.01 0.1 0.1 õ 0.01 0.1–0.1 0.1–1.01 õ 0.01–0.01–0.3 0.3 0.1 0.1–0.01–0.1 0.1 0.1–1.0 0.01–0.01–0.01–0.1 177 Sequence VLSAADKGNVKAAWGKa VLSAADKGNVKAAWG VLSAADKGNVKAAa VLSAADKGNVKA LSAADKGNVKAA SAADKGNVa KVGGHAAEYGAEAa KVGGHAAEYGAE KVGGHAAEYGA VGGHAAEYGAEALa VGGHAAEYGAEAa AEYGAELa AEYGAE GAEALERa AEALERM EALERM EALEa LSFPTTK DLSHGSAQV ALTKA LPGALSELS LPGALSEa LPGA LASHLPSDFTPAV ASHLPSDFTPAVHA ASHLPSDFTPAVH ASHLPSDFTPAV ASHLPSDFTPA ASHLPSDF ASHLPS ASHLP LPSDFTPAVH LPSDF LDKFLA MLTAEEKAAVTa MLTAEEKAAVa MLTAEEKAAa MLTAEEKA MLTAE LTAEEKA AEEKAA GKVKVDEVGGEALGRLa VKVDEVGGEALGRL DEVGGEALGR EVGGEALGRLa EVGGEALGR EALG ALG SNGMKGLDDLK KLHVDPEa ARNFGKFFa NFGKEFTPV VLQA a Fragment a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a a b b b b b b b b b b b b b b b b b b b 1–16 1–15 1–13 1–12 2–13 3–10 16–28 16–27 16–26 17–29 17–28 22–29 22–28 25–31 26–32 27–32 27–30 34–40 47–55 65–69 76–84 76–82 76–79 109–121 110–123 110–122 110–121 110–120 110–117 110–115 110–114 113–122 113–117 125–130 2–12 2–11 2–10 2–9 2–6 3–9 5–10 16–31 18–31 21–30 22–31 22–30 26–29 27–29 72–82 95–101 115–122 117–125 125–128 These peptides display hemopoietic activity.3 0.1 0.01–0.1 0.1 0.01–0.0 0.Hemoglobin as a Source of Peptides Table VIII Hemoglobin Fragments Isolated from Bovine Red Bone Marrow30 Content (nmol/g Tissue) õ 0.01–0.1–0.01–0.1 0.1 õ 0.1 0.01 0.01–0.01–0.1 0.1 õ 0.1 0.1 0.1–0.1–0.1 0.01–0.1 0.1 0.3 0.3 0.1–1.41 .1 0.3 0.1 0.01–0.1 0.1 0.01–0.01–0.0 0.1 0.01–0.01–0.1–1.01–0.1 0.1 0.01–0.01 0.01–0.01 0.1 0.01–0.1–0.01–0.3 0.01–0.01–0.1–0.01–0.1 õ 0.01–0.01–0.1 0.01–0.1 0.01–0.1 õ 0.01–0.
giving rise to ‘‘ladders’’ of stepwise C. part of that FIGURE 1 Hemoglobin sequences covered by peptides isolated from animal tissues and biological activities associated with respective sequences. OP—opioid. The products of both primary and secondary proteolytic attack are degraded by carboxy.and aminopeptidases. CC—coronaro-constrictory. regardless of the chosen species and tissue. (85)Phe-rrr (Ala/Thr/Lys/Ser/His)(87). IM—immunomodulatory. stepwise manner of hemoglobin degradation..178 Ivanov et al. NK—peptides from neokyotorphin group. Sequences covered by isolated peptides are marked by dark shading.and N-terminally shortened peptides. The obtained polypeptides are further cut into several smaller pieces. e. These positions for a-globin are (33)Met-Phe-(Leu/Ala)(35). HR— hormone releasing. (30)Arg-Leu-Leu-Val-Val(34). (69)Ala-rrr-(Leu/Met/Val)(76). As seen from the positions of the nonoverlapping sequences. giving rise to 2–4 fragments 30–60 amino acid residues long.( Leu / Ile ) -Val. . However. AG—antigonadotropic.( Val / Cys /Met ) -Val(Leu/Met)-(Ala/Gly)(115). (54)(Val/Ile)-rrr-(Ser/Asn)(72). The products of carboxypeptidase action are more frequently represented in Figures 4 and 5. 1992–1996) Fragment a a a a a a a a a a b b b b b b Table IX Sequence Source Human cerebellum Human cerebellum Rat hippocampus Human cerebellum Human cerebellum Human cerebellum Rat hippocampus Human cerebellum Human cerebellum Rat hippocampus Human prefrontal cortex Rat hippocampus Human cerebellum Human postcentral gyrus Human cerebellum Human cerebellum Pathology Alzheimer’s disease43 Healthy donor44 Ischemia45 Alzheimer’s disease43 Alzheimer’s disease43 Alzheimer’s disease43 Ischemia45 Alzheimer’s disease43 Alzheimer’s disease43 Ischemia45 Alzheimer’s disease45 Ischemia45 Alzheimer’s disease43 Alzheimer’s disease45 Alzheimer’s disease43 Alzheimer’s disease43 VLSPADKTNVKAAWGKVGAHAGEYGA-? VLSPADK-? ?-MFAAFPTTK-? FLSFPTTKTYFPHFDLSHGSAQV-? FLSFPTTKTYFPHFDLSH-? FLSFPTTK-? ?-TYFNHIDVSP-? LVTLAAHLP-? AAHLPAEFTP-? ?-LASVSTVLT-? VHLTPEEKSAVTAL-? ?-VVYPWTQRY-? FESFGDLSTPDAV-? (AV)MGNPK-? SELHCDKLHV—EFTPPVQAAYQK-? VCVLAHHFGKEFTPPVQAAY-? 1–26? 1–7? (32–40)? 33–55? 33–50? 33–40? (41–50)? 106–114? 110–119? (129–137)? 1–14? (33–41)? 42–54? 53–59? 89–132? 111–130? The obtained overall picture clearly speaks of a nonrandom.g. at sites (13)(Ala/Cys)Trp-(Gly/Glu)-Lys-(Val/Ile)(17). and (13) ( Ala/ Gly/ Thr)-( Phe/ Leu/ Ala )-Trp-Gly-Lys-Val (18). Hemoglobin Fragments as Peptide Markers of Brain Pathologies (Sleemon et al. or (127)Gln-Ala(Asp/Ala)-(Phe/Tyr)-Gln(131) in the b-globin. or (135) Val-Leu-Thr-Ser(138) in the a-globin. (88)Ala-rrrLeu(101) and (105)Leu-Leu(106). Types of biological activities: HP—hemopoietic. (127)Lys-PheLeu-(Ala/Ser)-(Asn/Ser)-Val-Ser(133). (108)Asn( Val /Met ) . the primary splitting might occur at the sites marked in Figures 4 and 5 by arrows. (55)Val-rrr-Ala(65). For b-globin the respective sites are (41)(Phe/Tyr)-Phe(42).
The obtained sequences are given in Tables X and XI at respective numbers. All splitting sites contain hydrophobic residues. as shown in the ﬁgure caption. (B) the supernatant of primary culture of human erythrocytes.Hemoglobin as a Source of Peptides 179 (Ala/Leu/Phe)-Trp. The collected fractions are underlined. As for the secondary splitting. . (A) The lysate of human erythrocytes.56 AU). the a-chain (32– 34) segment (Met-Phe-Leu) is cut at Met-Phe and Phe-Leu sites and the b-chain (14–17) segment [(Ala/Leu/Phe)-Trp-Gly-Lys] is apparently cut at FIGURE 3 RP-HPLC of the fractions obtained after size-exclusion separation. although FIGURE 2 Size-exclusion chromatography of peptide material from the lysate of human erythrocytes (A) and the supernatant of the primary culture of human erythrocytes (B). Absorbance range in mV (1800 mV Å 2. We tried to correlate the putative positions of primary cutting with the secondary structure of globin chains around those positions as well as their availability to intermolecular interactions with proteases. more work should be done to conﬁrm the above speculation since possible participation of more than one enzyme in degradation as well as inclusion into Figures 4 and 5 of peptides from various tissues and various species can serve as alternative explanations. For instance. Such diffuse speciﬁcity might be also associated with conformational features of the products of primary splitting. it is worth mentioning that it takes place at several adjacent peptide bonds rather than at strictly deﬁned single pairs of amino acid residues. Following the arguments given in previous section. we assume that primary proteolysis of hemoglobin takes place exclusively inside the erythrocytes while all the following steps predominantly occur on crossing the erythrocyte membrane and later within the tissue. There are no obvious regularities in amino acid sequences around the primary or secondary splitting sites. It is only clear that trypsin-like proteases do not participate in the processing of hemoglobin. It is clear from a brief glance at the picture that all loops. The peaks containing peptides subjected to sequenation are marked with numbers. Trp-Gly. abundance might be due to increasing uncertainty in identiﬁcation of the last C-terminal residues in the course of automatic sequence analysis by the Edman procedure. Certainly. and Gly-Lys sites. since there are no arginines and very few lysines in those sequences. Figure 6 presents the space ﬁlling model of the tetrameric hemoglobin globule in which helixconnecting loops of the polypeptide chains are marked with different colors and labeled with numbers.
06–0. seem to satisfy the steric requirements.05–0.05 0.and b-globins whose content in the equilibrium is ca. Such splitting explains the formation of the peptides a-chain (1–33) 15 and b-chain (1–41). Table XI Hemoglobin Fragments Released from Human Erythrocytes in Primary Culture Content (nmol/mL Cells) 0.02 0. leading to formation of additional degradation sites. the symmetric a-chain (69–76) and b-chain (56–72) ones.180 Ivanov et al. 60 amino acid residues. and b-chain (85–87) sites fall into the middle of respective helical segments. a-chain (88–101). belonging respectively to loops 3 and 8. Secondary structures of these chains are shown in Figure 7. a-chain (33–34) and b-chain (41–42)] become a plausible target for the protease. Besides the tetramer. more detailed analysis proves that the above-mentioned sites a-chain (33–35).12 No. One should also consider that conformational rearrangement might accompany the nicking of both the tetramer and the dissociated forms.08 0. as well as the adjacent a-helical amino acid residues [in particular.07 0.05 0.05 0.01–0.03–0.001– 0.05 0. The remaining achain (59–65). the concrete sites of primary splitting require identiﬁcation of longer peptides containing ca. 6 5 3 4 7 9 1 2 8 Sequence VLSPADAKTNAVKAWGKVGAHAGEYGAEALERMF VLSPADAKTNAVKAWGKVGAHAGEYGAEALERMa VLSPADAKTNAVKAWGKVGAHAGEYGAEALERa VLSPADAKTNAVKAWGKVGAHAGEYGAEALE VTLAAHLPAEGFTPAVHASLDKFLASVSTVL AAHLPAEGFTPAVHASLDKFLASVSTVLTSKYR TSKYR TSKY AHHFGKEFTPPVQAAYQKVVAGVANALAHRYH Fragment a a a a a a a a b 1–33 1–32 1–31 1–30 107–136 110–141 137–141 137–140 115–146 a The content of these peptides was 5–15 times higher in the blood samples of patients with Hodgkin’s disease than those of healthy donors. are represented at the surface of the globule.03–0.03–0. a-chain (105–107). 13 6 5 3 4 8 2 1 10 9 11 7 12 Sequence VLSPADKTNVKAAWGKV VLSPADKTNVKA VLSPADKTNV VLSPADKTN SDLHA VHLTPEEKSAV VHLTPEEKSA VHLTPEEK VYPWTQ VYPW SDGLAHLDNLKGTF TLSEL VVAGVANALAHRYH Fragment a a a a a b b b b b b b b 1–17 1–12 1–10 1–9 84–88 1–11 1–10 1–8 34–39 34–37 72–84 87–91 133–146 .06 0.04–0.10–0.03–0. Peptides Isolated from Human Erythrolysate15 Content (nmol/mL cells) 140–160 6–12 3–5 4–8 25–50 20–40 2–4 2–4 25–50 Table X No. In that case sites a-chain (36–52) and b-chain (35–57). However.06–0.26 It is too early to give a preference to either of the proposed degradation pathways. 0.03–0.10–0. if general principles of hemoglobin proteolysis leading to formation of peptide families are reasonably established. and b-chain (41–42) are not sterically accessible. monomeric a.05 0.03–0.08 0. referring to loops 2 and 7. In other words. Of the remaining sites.12 0.01% of the total protein 48 can also be a subject of enzymatic degradation.05 0.47 to a varying degree.
Sequences of a-globins: ( A ) bovine. ( B ) human. Sequences of biologically active peptides are printed in bold italic. ( C ) pig. . The primary splitting sites are marked by arrows. ( D ) Yakut ground squirrels Citellus undulatus.Hemoglobin as a Source of Peptides 181 FIGURE 4 a-Globin fragments isolated from different sources. and ( E ) rat.
182 Ivanov et al. (D) Yakut ground squirrels Citellus undulatus. . (C) pig. (B) human. FIGURE 5 b-Globin fragments isolated from different sources. Sequences of b-globins: (A) bovine. The primary splitting sites are marked by arrows. Sequences of biologically active peptides are printed in bold italic. and (E) rat.
(5) fragment 119–123]. (A) front view. That concept originally formulated in 1992 was further supported by the . (4) fragment 92–94. respectively. In other words. (7) fragment 35–50. respectively. we believe participation of hemoglobin fragments in homeostasis provides an example of a novel peptide-mediated regulatory pathway complementary to traditional hormonal or neuropeptide mechanisms. Loops of two a-chains are shown in light and dark blue [position numbers: (1) fragment 18–19. (9) fragment 96–99.and b-chains are shown in white and magenta.Hemoglobin as a Source of Peptides 183 FIGURE 6 Space-ﬁlling model of human deoxyhemoglobin tetrameric structure. (3) fragment 74–75. (8) fragment 77–80. (5) fragment 113–118] and those of two b-chains—in red and brown [position numbers: (6) fragment 18–19. (2) fragment 36–52. Helices of two a. (B) and (C) views of the model rotated by 90 around X and Y axes. HEMOGLOBIN FRAGMENTS AS COMPONENTS OF TISSUE-SPECIFIC PEPTIDE POOL The broad spectrum of biological effects exhibited by endogenous hemoglobin-derived peptides forces a conclusion that proteolytic degradation of hemo- globin carries out an important in vivo regulatory function.
The a-helices and loops are shown in magenta and yellow. (B) b-chain. (A) a-chain. We suggest that . FIGURE 7 A view of the overall structure fold of human deoxyhemoglobin. The numbers indicate the beginning and the end of the a-helical secondary structure elements. respectively. above-described data on intensive formation in and release from the erythrocytes of active peptides subsequently found in various tissues.30 It is generally accepted that the proteins normally present in the tissue are digested by proteolytic enzymes after fulﬁlling their function.184 Ivanov et al.
which are further utilized in metabolic reactions. However.1–10 Seconds–minutes Transmission of nerve impulse Minutes–hours Regulation of physiological processes in the tissue or the whole organism Fragments of functional proteins Functional protein Action of tissue proteinases 10–10. we suggest that the tissue-speciﬁc peptide pool predominantly controls long-term processes. a term proposed in early 80-ies for the components with low molecular mass fractions of the total tissue extracts. and cell death. The latter includes cell proliferation. . As a result of that process.000 Hours–days Maintenance of tissue homeostasis protein elimination does not occur by random hydrolysis directly leading to amino acids.. poorly characterized mixtures containing all types of regulatory peptides presented in Table XII. and VII) provide a good example of that tendency. Similarly to the peptide pool.’’ ‘‘peptide background.’’ or more accurately as ‘‘tissue-speciﬁc peptide pool. Hemorphins and closely related peptides with their opioid receptor binding ability consistently found in most of the investigated sources (see Tables I. is responsible for tissue homeostasis. Instead. Alzheimer’s disease. 43. However.001–1.’’ The properties of that pool depend upon the concrete set of peptide components as well on individual levels of those components. a large number of peptides is formed that can be deﬁned as a ‘‘peptide buffer.000 Alteration of the level in the tissue Binding to receptors of homologous hormones 100–10. it represents a speciﬁc process regulated by the level of tissue-speciﬁc proteases and the availability of their substrates. such as human lung adenocarcinoma 14 (Tables II). the signal peptide molecules of the nervous tissue (neurotransmitters and/or neurohormones) or endocrine system (hormones and parahormones) are released from a narrow circle of speciﬁc precursors for which no other than having a precursor function is known.0 0.34 and regulate proliferation and differentiation of normal cells 38. all the above-mentioned pathologies have a common principle feature — they involve changes in tissue homeostasis or metabolic state of the cells.Hemoglobin as a Source of Peptides Table XII Distinctive Features of Peptidergic Regulatory Systems Peptidergic Regulatory System Characteristic Peptides Precursor Type of processing Level (pM/g tissue) Type of regulation Mechanism of action Nervous Endocrine and Paracrine Tissue-Speciﬁc Peptide Pool 185 Receptor binding constants (Kd . IV. Components of the tissue speciﬁc peptide pools have several features in common with the so called ‘‘cytomedines’’. Since the intensity of proteolytic processes depends on such a relatively conservative parameter as the metabolic state of the organism.41 provides a convincing argument favoring our view. There is a growing evidence of changes in tissue composition of hemoglobin-derived peptides accompanying various pathologies.45 brain ischemia 45 (Table IX).001–1. cytomedines were ascribed a homeostatic role for the given tissue (see the review49 and the references therein). The binding afﬁnities of the pool peptides are by several orders of magnitude lower than for hormones or neurotransmitters. V. most of the porperties of cytomedines were studied on complex. Prevention of cell transformation and lysis of tumor cells also fall into that category. The recent ﬁnding that hemoglobin fragments induce death of transformed cells 13.e. Notwithstanding the clinical and biochemical differences. Components of the peptide pool bind to the same receptors as neurotransmitters or hormones modulating thereby the availability of the receptors to their ‘‘true’’ ligands. nM) Time range of action Biological role Neurotransmitters Hormones Speciﬁc protein precursor Discrete site speciﬁc processing 0. In contrast to peptides derived from functional proteins. whatever the origin of the disease: cell transformation (adenocarcinoma). differentiation. Characteristic properties of peptides from the tissue-speciﬁc pool in comparison with the classical regulatory peptides are summarized in Table XII. and Hodgkin’s disease 47 (Table X). these peptides are found in higher amounts and typically occur as families of related molecules rather than single representatives. i.0 Synaptic secretion Extracellular secretion Binding to receptors Binding to receptors in in postsynaptic cellular membranes membrane 1–1000 0.
0 kDa molecular masses about (Figure 2) were collected and lyophilized. identiﬁcation of active peptides derived in vivo from cytochrome c oxidase.1% solution of triﬂouroacetic acid (TFA) in water and subjected to separation on Nucleosil 120/5m C8 cartridge (4. 56 and other functional proteins 30. 99% of erythrocytes) was washed four times by buffer A (0. AB(IV).1% TFA in water). In that case the function of erythrocytes might be compared with the function of endocrine gland. Destruction of Erythrocytes The washed clean cells were subjected to two cycles of freezing ( 070 C) and unfreezing (5 C).2). A portion of lyophilized supernatant obtained from 50 mL of blood cells was dissolved in 8 mL 0. Amino Acid Sequences Amino acid sequences of isolated peptides were determined in the gas-phase sequencer Applied Biosystems . 55 ﬁbrinogen.25 mL of supernatant was dissolved in 10 mL 0. and the whole organism. The amount of 0. Masherey-Nagel. then the elution was performed for 60 min by linear gradient of acetonitril from 0 to 60% of buffer B (0. The cells were separated from plasma by centrifugation at 1000 rpm for 15 min at 0 C. homogenized in Potter S (B.52 – 54 albumins. Both lysate and supernatant were prepared from the same batch of cells.1M acetic acid. The obtained supernatants were collected and lyophilized. On the contrary. aged 20–35 years] by veinpuncture after conﬁrmation of their health status by Research Haematological Centre. Elution was carried out at 120 mL/h. Size-Exclusion Chromatography The samples were separated on Sephadex G-25 sf (Pharmacia. the cells were pelleted by centrifugation in conditions described above.1M acetic acid and injected into the size-exclusion column. 80% acetonitril solution in water) at 0. 30. MATERIALS AND METHODS Preparation of Human Erythrocytes Peripheral venous blood was obtained from 10 healthy volunteers [7 men and 3 women with different blood groups A(II). Supernatant of the Primary Culture of Erythrocytes Ten milliliters of washed cells were incubated with 40 mL of buffer A in culture ﬂacks for 4 h at 37 C. it is only one of the components (although an important one) of the general system of peptidergic regulation of tissue homeostasis.0 1 250 mm. 51 immunoglobulins. several organs.025M NaH2PO4 containing 0. pH 7. One can even suggest that this system is phylogenetically more ancient than the endocrine and the nervous system since the latter deal with higher level of regulation. Endogenous fragmentation of the hemoglobin and the properties of respective peptides are studied much better than analogous processes with other functional proteins. 1 15 min) at Biofuge B (Heraeus. More data should be accumulated before the concept of tissue-speciﬁc peptide pool ﬁnds its ﬁnal shape. Sweden) column (2. equilibrated with buffer A (0. B(III). However. isolated phenomenon.1M NaCl. Supernatant was immediately injected into the sizeexclusion column. The column was washed for 5 min by buffer A.5 1 85 cm) equilibrated with 0.186 Ivanov et al. The obtained pellet (10–12 billions of cells. with detection at wavelengths 206/280 nm. Although it is not known whether the abovementioned changes in peptide composition are the cause or the consequence of the disease. these changes are in full accord with our views of the biological role of tissue-speciﬁc peptide pool.0 1 250 mm) in linear acetonitril gradient from 8 to 40%. Germany). Russian Academy of Medical Sciences. Germany) (900 rev 1 5 min) and centrifuged (10000 rev.57 allows us to conclude that the regulatory role of hemoglobin-derived peptides should not be considered a unique. or impaired lymphoproliferation (Hodgkin’s disease). involving several types of tissues.75 mL/min. Isolation of the Peptides The peaks corresponding to homogeneous substances were collected as shown in Figure 3. The detection was carried out at 226 nm.25%).1M acetic acid and subjected to centrifugation at 11000 rpm for 5 min. tissue atrophy (Alzheimer’s disease and ischemia).5–4. Braun.1% TFA. Blood samples (25 mL) were placed into the tubes containing citrate buffer (to ﬁnal citrate concentration of 0. The fractions corresponding to the zone of elution of substances with 0. RP-HPLC Separation The material obtained after size-exclusion chromatography was dissolved in 250 mL of 0. Germany). regardless of these developments there is little doubt that besides the oxygen transport (and possibly the transport of nitrogen oxide 50 ) hemoglobin serves as a rich source of bioactive peptides. After incubation. lyophilized and subjected to separation on Nucleosil 120/5m C18 cartridge (4. Still.
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