Methods of structural mass spectrometry have become more popular to study protein structure and dynamics. Among them, fast photochemical oxidation of proteins (FPOP) has several advantages such as irreversibility of modifications and more facile determination of the site of modification with single residue resolution. In the present study, FPOP analysis was applied to study the hemoglobin (Hb) - haptoglobin (Hp) complex allowing identification of respective regions altered upon the complex formation. FPOP footprinting using a timsTOF Pro mass spectrometer revealed structural information for 84 and 76 residues in Hp and Hb, respectively, including statistically significant differences in the modification extent below 0.3%. The most affected residues upon complex formation were Met76 and Tyr140 in Hbα, and Tyr280 and Trp284 in Hpβ. The data allowed determination of amino acids directly involved in Hb - Hp interactions and those located outside of the interaction interface yet affected by the complex formation. Also, previously modeled interaction between Hb βTrp37 and Hp βPhe292 was not confirmed by our data. Data are available via ProteomeXchange with identifier PXD021621.

• MeSH
• aminokyseliny chemie   metabolismus   MeSH
• footprinting proteinů metody   MeSH
• haptoglobiny chemie   metabolismus   MeSH
• hemoglobiny chemie   metabolismus   MeSH
• hmotnostní spektrometrie metody   MeSH
• hydroxylový radikál chemie   MeSH
• lidé MeSH
• molekulární modely MeSH
• molekulární struktura MeSH
• oxidace-redukce MeSH
• vazba proteinů MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

A recombinant plasmid containing a 6.5-kb fragment of nontypeable Haemophilus influenzae (NTHI) chromosomal DNA was shown to confer a hemoglobin-haptoglobin-binding phenotype on Escherichia coli. Use of a mini-Tn10kan transposon for random insertion mutagenesis of this recombinant plasmid allowed localization of the NTHI DNA responsible for this hemoglobin-haptoglobin-binding phenotype to a 3.5-kb PstI-XhoI fragment within the 6.5-kb NTHI DNA insert. When this mutagenized NTHI DNA fragment was used to transform the wild-type NTHI strain, the resultant kanamycin-resistant mutant exhibited significantly decreased abilities to bind hemoglobin-haptoglobin and utilize it as a source of heme for aerobic growth in vitro. This mutant also lacked expression of a 115-kDa outer membrane protein that was present in the wild-type parent strain. Transformation of this mutant with wild-type NTHI chromosomal DNA restored the abilities to bind and utilize hemoglobin-haptoglobin and to express the 115-kDa outer membrane protein. Nucleotide sequence analysis of the relevant NTHI DNA revealed the presence of a gene, designated hhuA, that encoded a predicted 117,145-Da protein. The HhuA protein exhibited features typical of a TonB-dependent outer membrane receptor and had significant identity with the hemoglobin receptors of both Haemophilus ducreyi and Neisseria meningitidis.

• MeSH
• bakteriální geny MeSH
• DNA bakterií genetika   MeSH
• Haemophilus influenzae genetika   metabolismus   MeSH
• haptoglobiny * metabolismus   MeSH
• hem metabolismus   MeSH
• hemoglobiny * metabolismus   MeSH
• inzerční mutageneze MeSH
• klonování DNA MeSH
• molekulární sekvence - údaje MeSH
• proteiny vnější bakteriální membrány genetika   metabolismus   MeSH
• restrikční mapování MeSH
• sekvence aminokyselin MeSH
• sekvence nukleotidů MeSH
• sekvenční homologie aminokyselin MeSH
• sekvenční seřazení MeSH
• Publikační typ
• Research Support, U.S. Gov't, P.H.S. MeSH
• srovnávací studie MeSH

BACKGROUND: The scavenger receptor for complexes hemoglobin-haptoglobin (CD163), which is expressed on monocytes/ macrophages, is shed to the body fluids in a soluble form (sCD163). OBJECTIVES: To evaluate the dynamics of sCD163 in the blood of patients undergoing cardiac surgery. PATIENTS AND METHODS: Sixty-one adult patients who underwent coronary artery bypass grafting (CABG) were enrolled in the study. They were assigned to undergo CABG using either cardiopulmonary bypass (CPB), "on-pump", (22 patients), modified CPB, mini "on-pump", (17 patients) or without CPB, "off-pump", (22 patients) surgery. Serum levels of sCD163 in venous blood samples taken before and after surgery, and during an early postoperative period, were evaluated by Macro 163(TM) diagnostic kit (IQ Products, Groningen, NL). RESULTS: Compared to the preoperative levels ("on-pump"; 344 ng/mL, "off-pump"; 314.5 ng/mL, mini-invasive "on-pump"; 336.5 ng/mL) serum levels were elevated at the finish of surgery, reaching maximum at the 1(st) postoperative day ("onpump"; 658 ng/mL; p<0.05, "off-pump"; 810.5 ng/mL; p<0.01; mini-invasive "on-pump"; 663 ng/mL; non-significant).No significant differences regarding the serum levels of sCD163 between different surgical approaches were found. CONCLUSION: Serum level of sCD163 scavenger molecule for hemoglobin is elevated at the end of surgery and at the 1(st) postoperative day, being little influenced by cardiopulmonary bypass.

• MeSH
• antigeny diferenciační myelomonocytární krev   MeSH
• CD antigeny krev   MeSH
• haptoglobiny metabolismus   MeSH
• hemoglobiny metabolismus   MeSH
• kardiopulmonální bypass škodlivé účinky   MeSH
• koronární bypass škodlivé účinky   MeSH
• lidé středního věku MeSH
• lidé MeSH
• receptory buněčného povrchu krev   MeSH
• senioři MeSH
• zánět etiologie   krev   MeSH
• Check Tag
• lidé středního věku MeSH
• lidé MeSH
• mužské pohlaví MeSH
• senioři MeSH
• ženské pohlaví MeSH

Haptoglobin is a plasma protein of mammals that plays a crucial role in vascular homeostasis by binding free haemoglobin released from ruptured red blood cells. Trypanosoma brucei can exploit this by internalising haptoglobin-haemoglobin complex to acquire host haem. Here, we investigated the impact of haptoglobin deficiency (Hp-/-) on T. brucei brucei infection and the parasite ́s capacity to internalise haemoglobin in a Hp-/- mouse model. The infected Hp-/- mice exhibited normal disease progression, with minimal weight loss and no apparent organ pathology, similarly to control mice. While the proteomic profile of mouse sera significantly changed in response to T. b. brucei, no differences in the infection response markers of blood plasma between Hp-/- and control Black mice were observed. Similarly, very few quantitative differences were observed between the proteomes of parasites harvested from Hp-/- and Black mice, including both endogenous proteins and internalised host proteins. While haptoglobin was indeed absent from parasites isolated from Hp-/-mice, haemoglobin peptides were unexpectedly detected in parasites from both Hp-/- and Black mice. Combined, the data support the dispensability of haptoglobin for haemoglobin internalisation by T. b. brucei during infection in mice. Since the trypanosomes knock-outs for their haptoglobin-haemoglobin receptor (HpHbR) internalised significantly less haemoglobin from Hp-/- mice compared to those isolated from Black mice, it suggests that T. b. brucei employs also an HpHbR-independent haptoglobin-mediated mode for haemoglobin internalisation. Our study reveals a so-far hidden flexibility of haemoglobin acquisition by T. b. brucei and offers novel insights into alternative haemoglobin uptake pathways.

• MeSH
• haptoglobiny * genetika   metabolismus   MeSH
• hemoglobiny * metabolismus   MeSH
• modely nemocí na zvířatech MeSH
• myši inbrední C57BL MeSH
• myši knockoutované * MeSH
• myši MeSH
• proteomika metody   MeSH
• Trypanosoma brucei brucei * metabolismus   MeSH
• trypanozomóza africká * parazitologie   imunologie   MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

Ischemicko-reperfuzní procesy provázející akutní koronární syndromy jsou provázeny významnou aktivací imunitního systému. Molekula CD163 patří mezi významné molekuly scavengerového systému, které se podílejí na eliminaci hemoglobin-haptoglobinového komplexu a mají tak potenciál snížit produkci reaktivních kyslíkových radikálů. Cílem studie bylo sledovat změny hladin sCD163 v průběhu 96 hodin akutního koronárního syndromu léčeného direktní perkutánní intervencí. V souboru 29 pacientů (25 mužů, 4 žen) průměrného věku 64,55 let ± 7,55 let byly stanoveny hladiny sCD163 v době přijetí, následně za 24 a 96 hodin. Studie prokázala významné zvýšení hladiny sCD163 již v čase přijetí a v dalších měřeních se dále zvyšovala [čas 0 hod: 1770,5 (1451; 2096,25) vs. 1178 (1078; 1265) μg/l, p < 0,001, 24 hod.: 2063 (1589; 2453) vs. 1178 (1078; 1265) μg/l, p < 0,001, 96 hod: 2116 (1924; 2458) vs. 1178 (1078; 1265) μg/l, p < 0,001]. Analýza vztahu k funkci levé komory (ejekční frakci) prokázala statisticky významnou asociaci ve všech sledovaných obdobích. Studie ukázala na potenciální význam sCD163 pro stratifikaci rizika těchto nemocných.

Background: The ischemia/reperfusion process in the course of acute coronary syndrome with ST-segment elevation is associatedwith significant increase of immune system activation. CD163 molecule belongs to important parts of the scavenger system,which helps to eliminate hemoglobin-haptoglobin complex and has the potential to suppress reactive oxygen radicals production. Aim: The aim of our study was to assess sCD163 level in the course of STEMI.Patients and method: Plasma sDC163 level was assessed in a group of 29 patients (25 males, 4 females) of average age 64.5 let± 7.5 yrs. at the time of admission, at 24 hours and at 96 hours. Results: The plasma sCD163 level was increasing during the time of observation [0h: 1770.5 (1451; 2096.25) μg/L vs. 1178 (1078;1265) μg/L, p < 0.001, 24h: 2063 (1589; 2453) vs. 1178 (1078;1265) μg/L, p < 0.001, 96h: 2116 (1924; 2458) vs. 1178 (1078; 1265) μg/L, p < 0.001]. Plasma sCD163 level was associated with leftventricle ejection fraction. Conclusion: The study showed a potential significance of sCD163 assessment as a prognostic marker.

• MeSH
• biologické markery krev   MeSH
• CD antigeny * krev   MeSH
• humorální imunita MeSH
• infarkt myokardu s elevacemi ST úseků * MeSH
• lidé středního věku MeSH
• lidé MeSH
• reperfuze myokardu MeSH
• scavengerové receptory MeSH
• senioři MeSH
• Check Tag
• lidé středního věku MeSH
• lidé MeSH
• mužské pohlaví MeSH
• senioři MeSH
• ženské pohlaví MeSH
• Publikační typ
• klinická studie MeSH
• práce podpořená grantem MeSH