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Molecular cloning and characterization of a complement-depleting factor from king cobra, Ophiophagus hannah
Lin Zeng1, Qian-Yun Sun, Yang Jin
1Key Laboratory of Animal Models and Human Disease Mechanisms, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming 650223, Yunnan, China.
Insights
Cobra venom factor (CVF) from Ophiophagus hannah (OVF) was purified and characterized. OVF exhibits unique structural features, including distinct N-terminal sequences and glycosylation patterns, offering insights into CVF function.
Area of Science:
- Biochemistry
- Immunology
- Molecular Biology
Background:
- Cobra venom factor (CVF) is a potent anti-complement protein found in snake venom.
- While CVF has been isolated from various cobra species, full-length cDNA sequences are scarce.
- Understanding CVF structure is crucial for elucidating its interaction with the complement system.
Purpose of the Study:
- To purify and characterize a novel cobra venom factor (OVF) from Ophiophagus hannah.
- To determine the molecular weight, subunit composition, and anti-complement activity of OVF.
- To clone and analyze the cDNA sequence of OVF, comparing it with known CVFs.
Main Methods:
- Purification using gel filtration, ion-exchange, and heparin affinity chromatography.
- SDS-PAGE for molecular weight determination under reducing and non-reducing conditions.
- Reverse transcription-PCR and 5'-RACE for cDNA cloning; MALDI-TOF and protein sequencing for confirmation.
Main Results:
- OVF was purified to homogeneity with a molecular weight of 140 kDa, composed of α (72 kDa), β (45 kDa), and γ (32 kDa) chains.
- OVF demonstrated significant anti-complement activity (154 units/mg).
- Phylogenetic analysis indicated OVF is closely related to N. kaouthia CVF. Unique features include a distinct OVF γ chain N-terminus, a single N-linked glycosylation site on the α chain, and an additional cysteine residue in the γ chain.
Conclusions:
- OVF represents a unique variant of cobra venom factor with distinct structural characteristics.
- The identified unique features of OVF may play a role in its structure-function relationship with the complement system.
- Further investigation into these specific characteristics can enhance our understanding of CVF-complement interactions.
Abstract:
Cobra venom factor (CVF) is an anti-complement factor existing in cobra venom. CVF proteins have been purified from the venoms of Naja haje, Naja siamensis, Naja atra, Naja kaouthia, Naja naja, Naja melanoleuca and Austrelaps superbus, but only three full-length cDNA sequences of CVF are available. In the present work, a cobra venom factor termed OVF was purified from the crude venom of Ophiophagus hannah by successive gel filtration, ion-exchange and heparin affinity chromatography steps. The purified OVF was homogenous on the SDS-PAGE gel with an apparent molecular weight of 140 kDa under non-reducing conditions. Under reducing conditions, OVF was divided into three bands with apparent molecular weight of 72 kDa (α chain), 45 kDa (β chain) and 32 kDa (γ chain), respectively. OVF consumed complement components with anti-complement activity of 154 units per mg. By using Reverse transcription-PCR and 5'-RACE assay, the open reading frame of OVF was obtained. MALDI-TOF and protein sequencing assays confirmed the cloned cDNA coding for OVF protein. The cDNA sequence of OVF is conservative when aligned with that of other CVFs. Phylogenetic analysis revealed OVF is closer to CVF from N. kaouthia than to AVF-1 and AVF-2 from A. superbus. Our results demonstrated that OVF has its unique features as following: 1) The N-terminal amino acid sequence of OVF γ chain is different from that of other known CVFs, suggesting that the OVF γ chain might be further processed; 2) Unlike N. kaouthia CVF and A. superbus AVF-1, which have potential N-linked glycosylation sites located in both α and β chain, OVF only has N-linked glycosylation site in its α chain as revealed by Schiff's reagent staining and protein sequence analysis; 3) In addition to the 27 well conserved cysteine residues in all known CVFs, OVF have an additional cysteine residue in its γ chain. Understanding the importance of above mentioned specific characteristics might provide useful information on structure-function relationship between CVF and complement system.
