Organizing co mmittee

 
 

---

Poster presentation 
219 
RIBOSOME-INACTIVATED PROTEINS (RIPS) OF THE BLACK 
ELDER Sambucus nigra 
 
Kh.T. Agzamkhuzhaeva, Yu.I. Oshchepkova* 
 
A.S. Sadykov Institute of Bioorganic Chemistry Academy of Sciences of the Republic of 
Uzbekistan, *
e-mail: [e-mail] 
 
Ribosome-inactivating proteins (RIPs) belong to a class of enzymes found in plants, 
fungi, algae, and bacteria. RIPs exhibit N-β-glycosylase rRNA activity, which leads to 
cleavage of an adenine residue in the conserved 28S rRNA site. Cleavage of this single 
N-glycosidic bond is irreversible and interferes with association between elongation 
factors and the ribosome, causing inhibition of protein synthesis. This inactivation 
occurs by removing a specific adenine residue from the highly conserved (sarcin/ricin) 
loop of large ribosomal RNA. 
Sambucus, 
belonging to the Adoxaceae family and native 
to Europe, Asia, America and Africa, contains a complex mixture of different types of 
RIPs and related lectins.
 
The presence of RIP and lectins was studied mainly in 
Sambucus ebulus L.
(dwarf elderberry), 
Sambucus nigra L. 
(European elderberry), 
Sambucus sieboldiana Blume ex Graebn.
(Japanese elder) and 
Sambucus racemosa L. 
(red elder). 
The aim of this work is to study the composition of ribosome-inactivating proteins 
from the bark and berries of the black elderberry 
Sambucus nigra L
., growing on the 
territory of the Republic of Uzbekistan. 
The composition of ribosome-inactivating proteins in the bark and berries of black 
elderberry
Sambucus nigra L
., collected in the Botanical Garden of the Republic of 
Uzbekistan, was studied. To identify the isolated compounds on a Bruker MicroFlex 
spectrometer, a mass spectral MALDI-TOF of the protein composition obtained from 
the bark was carried out and proteins were found in the mass range of 61–63.0 kDa, 
corresponding in mass to RIP-2. The repeated mass spectral MALDI-TOF showed that 
incubation with DTT (50 mM) leads to the disappearance of the signal in the mass range 
of 61-63.0 kDa and an increase in the signal in the range of 30-32 kDa, which 
corresponds to the breakdown of RIP-2 into two subunits and indicates the disulfide 
nature of the isolated RIPs. According to HPLC-MS/MS sequencing, the presence of 9 
RIP-2 was found in the bark sample. Based on the obtained amino acid sequences using 
the CLUSTAL O (1.2.4) program, the amino acid sequence of the identified type 2 RIPs 
was compared and similar and conserved residues were identified, which will further 
help identify new RIPs. In the course of the studies, the tryptic peptide 
LSLVVLQMVSEAAR containing the RIP active site motif (EAAR) was identified 
with a high degree of probability. Mass spectral data on this fragment will allow further 
identification of other RIP-1/2 from other samples of natural origin. 2 RIPs were 
isolated from berries, one of which is in a reduced form containing one chain. 
Establishing the amino acid sequence in the isolated RIP from black elderberries with a 
molecular mass of 62337 Da confirmed our hypothesis of identification by the tryptic 
peptide containing the RIP active site (EAAR). 
Thus, in the course of the studies, a tryptic 
peptide containing the RIP active center motif (EAAR) was identified with a high degree of 
probability. Mass spectral data on this fragment will allow further identification of other RIP-
1/2 from other samples of natural origin. The obtained data of De novo sequencing of the 
LSLVVLQMVSEAAR peptide will allow identification of RIP from other sources. 

---

Poster presentation 
220