Structural and functional insights into lipid-bound nerve growth factors.

Nerve growth factor (NGF) is a dimeric molecule that modulates the survival, proliferation, and differentiation of nervous cells and is also known to act on cells of the immune system and endocrine system. NGFs extracted from mouse submaxillary gland and cobra venom have different immunological behaviors, yet the underlying mechanism remains unclear. Here we report the crystal structure of the NGF purified from Chinese cobra Naja naja atra (cNGF), which unexpectedly reveals a 2-tailed lipid molecule that is embedded between the two protomers of the NGF homodimer. In addition, crystallographic analysis indicated that the purified mouse NGF(mNGF) is free from lipid but can bind lysophosphatidylserine (lyso-PS) in the same pocket as cNGF. Bioassays indicated that the binding of lipid molecules to cNGF and mNGF are essential for their mast cell activation activity and abates their p75(NTR) binding capacity. Taken together, these results suggest a new mechanism for the regulation of the function of NGF.

Structural and functional analysis of natrin, a venom protein that targets various ion channels.

Cysteine-rich secretory proteins (CRISPs) are secreted single-chain proteins found in different sources. Natrin is a member of the CRISP family purified from the snake venom of Naja naja atra, which has been reported as a BKca channel blocker. In our study, crystals of natrin were obtained in two different crystal forms and the structure of one of them was solved at a resolution of 1.68A. Our electrophysiological experiments indicated that natrin can block the ion channel currents of the voltage-gated potassium channel Kv1.3. Docking analyses of the interaction between natrin and Kv1.3 revealed a novel interaction pattern different from the two previously reported K(+) channel inhibition models termed "functional dyad" and "basic ring". These findings offered new insights into the function of natrin and how the specific interactions between CRISPs and different ion channels can be achieved.

Structure of a cardiotoxic phospholipase A(2) from Ophiophagus hannah with the "pancreatic loop".

The crystal structure of an acidic phospholipase A(2) from Ophiophagus hannah (king cobra) has been determined by molecular replacement at 2.6-A resolution to a crystallographic R factor of 20.5% (R(free)=23.3%) with reasonable stereochemistry. The venom enzyme contains an unusual "pancreatic loop." The conformation of the loop is well defined and different from those in pancreas PLA(2), showing its structural variability. This analysis provides the first structure of a PLA(2)-type cardiotoxin. The sites related to the cardiotoxic and myotoxic activities are explored and the oligomer observed in the crystalline state is described.

Preliminary Crystallographic Analysis of Phospholipase A(2) from Naja naja kaouthia Lesson Venom.

An acidic phospholipase A(2) isolated from the venom of Naja naja kaouthia Lesson in Guangxi exhibits anticoagulative and hemolytic activities. In this work, the enzyme was crystallized by the method of hanging drop vapor diffusion. Two crystal forms were obtained and characterized by X-ray diffraction. One of them belonged to space group P 4 ( 3 ) 2 ( 1 )2 or P4(1)2(1)2 with unit cell parameters a b 8.797 nm, c 10.831 nm and there were three molecules per asymmetric unit the other belonged to space group P2(1)3 with unit cell parameters a b c 6.840 nm and there was one molecule per asymmetric unit. The diffraction data were collected up to 0.28 nm for each crystal form. The crystal properties of Naja naja verom phospholipase A(2) from different geographical regions are compared.

Cloning and Sequence Analysis of cDNAs Encoding Two Acidic PLA(2) from venom of Ophiophagus hannah(King Cobra), Guangxi Species.

Total RNA was extracted from venom glands of Ophiophagus hannah, Guangxi species. The cDNAs encoding PLA(2) were amplified by RT-PCR and cloned into the PUCm-T vector. The positive clones encoding two acidic PLA(2) (APLA(2)-1 and APLA(2)-2) were selected and bidirectionally sequenced. Their complete amino acid sequences were deduced and found to be identical to the known amino acid sequences. Their isoelectric points calculated by computer agreed with the values determined with their protein. Homology analysis indicated that the mature peptide of APLA(2)-1 had high homology with PLA(2) from venoms of Ophiophagus hannah, Fujian and Taiwan species, but APLA(2)-2 had lower homology. The most striking difference between APLA(2)-2 and other PLA(2) from Ophiophagus hannah venoms is the missing of a extra "pancreatic loop" at residues 62--66 in APLA(2)-2, and it may be related to their species evolution and biological activity.

NGF-Tf Conjugate Prevents Degeneration of Substantia Nigra Neurons in a Mouse Model of Parkinson's Disease.

Nerve growth factor(NGF) was purified from Naja naja atra snake venom and conjugated to transferrin(Tf). This conjugate was intravenously injected into a mouse model of Parkinson's disease (PD). Both immunohistochemical staining and pathological detection showed that the NGF-Tf conjugate could prevent the loss of tyrosine hydroxylase-immunoreactive neurons located in substantia nigra and the cell counts of NGF group were 2 330.0+/-260.3, and those of the MPTP group and the control group were 797.0+/-121.4 and 2 381.0+/-158.0, respectively. In addition, electron microscopic examination revealed significant protection against demyelination and vacuolation in subtantia nigra neurons in contrast to the control group. The i.v. injected NGF-Tf conjugate also reversed the neurodegenerative changes such as karyopyknosis, chromatolysis and intracytoplasmic inclusion in diseased neurons.