Tej P. Singh

Zinc α2-Glycoprotein: A Multidisciplinary Protein

Zinc α2-glycoprotein (ZAG) is a protein of interest because of its ability to play many important functions in the human body, including fertilization and lipid mobilization. After the discovery of this molecule, during the last 5 decades, various studies have been documented on its structure and functions, but still, it is considered as a protein with an unknown function. Its expression is regulated by glucocorticoids. Due to its high sequence homology with lipid-mobilizing factor and high expression in cancer cachexia, it is considered as a novel adipokine. On the other hand, structural organization and fold is similar to MHC class I antigen-presenting molecule; hence, ZAG may have a role in the expression of the immune response. The function of ZAG under physiologic and cancerous conditions remains mysterious but is considered as a tumor biomarker for various carcinomas. There are several unrelated functions that are attributed to ZAG, such as RNase activity, regulation of melanin production, hindering tumor proliferation, and transport of nephritic by-products. This article deals with the discussion of the major aspects of ZAG from its gene structure to function and metabolism. (Mol Cancer Res 2008;6(6):892–906)

Inhibition of protein aggregation: supramolecular assemblies of arginine hold the key.

Background Aggregation of unfolded proteins occurs mainly through the exposed hydrophobic surfaces. Any mechanism of inhibition of this aggregation should explain the prevention of these hydrophobic interactions. Though arginine is prevalently used as an aggregation suppressor, its mechanism of action is not clearly understood. We propose a mechanism based on the hydrophobic interactions of arginine. Methodology We have analyzed arginine solution for its hydrotropic effect by pyrene solubility and the presence of hydrophobic environment by 1-anilino-8-naphthalene sulfonic acid fluorescence. Mass spectroscopic analyses show that arginine forms molecular clusters in the gas phase and the cluster composition is dependent on the solution conditions. Light scattering studies indicate that arginine exists as clusters in solution. In the presence of arginine, the reverse phase chromatographic elution profile of Alzheimers amyloid beta 1-42 (Aβ1-42) peptide is modified. Changes in the hydrodynamic volume of Aβ1-42 in the presence of arginine measured by size exclusion chromatography show that arginine binds to Aβ1-42. Arginine increases the solubility of Aβ1-42 peptide in aqueous medium. It decreases the aggregation of Aβ1-42 as observed by atomic force microscopy. Conclusions Based on our experimental results we propose that molecular clusters of arginine in aqueous solutions display a hydrophobic surface by the alignment of its three methylene groups. The hydrophobic surfaces present on the proteins interact with the hydrophobic surface presented by the arginine clusters. The masking of hydrophobic surface inhibits protein-protein aggregation. This mechanism is also responsible for the hydrotropic effect of arginine on various compounds. It is also explained why other amino acids fail to inhibit the protein aggregation.

Enzymatic toxins from snake venom: structural characterization and mechanism of catalysis

Snake venoms are cocktails of enzymes and non‐enzymatic proteins used for both the immobilization and digestion of prey. The most common snake venom enzymes include acetylcholinesterases, l‐amino acid oxidases, serine proteinases, metalloproteinases and phospholipases A2. Higher catalytic efficiency, thermal stability and resistance to proteolysis make these enzymes attractive models for biochemists, enzymologists and structural biologists. Here, we review the structures of these enzymes and describe their structure‐based mechanisms of catalysis and inhibition. Some of the enzymes exist as protein complexes in the venom. Thus we also discuss the functional role of non‐enzymatic subunits and the pharmacological effects of such protein complexes. The structures of inhibitor–enzyme complexes provide ideal platforms for the design of potent inhibitors which are useful in the development of prototypes and lead compounds with potential therapeutic applications.

First Structural Evidence of a Specific Inhibition of Phospholipase A2 by alpha-Tocopherol (Vitamin E) and its Implications in Inflammation: Crystal Structure of the Complex Formed Between Phospholipase A2 and alpha-Tocopherol at 1.8 A Resolution

This is the first structural evidence of alpha-tocopherol (alpha-TP) as a possible candidate against inflammation, as it inhibits phospholipase A2 specifically and effectively. The crystal structure of the complex formed between Vipera russelli phospholipase A2 and alpha-tocopherol has been determined and refined to a resolution of 1.8 A. The structure contains two molecules, A and B, of phospholipase A2 in the asymmetric unit, together with one alpha-tocopherol molecule, which is bound specifically to one of them. The phospholipase A2 molecules interact extensively with each other in the crystalline state. The two molecules were found in a stable association in the solution state as well, thus indicating their inherent tendency to remain together as a structural unit, leading to significant functional implications. In the crystal structure, the most important difference between the conformations of two molecules as a result of their association pertains to the orientation of Trp31. It may be noted that Trp31 is located at the mouth of the hydrophobic channel that forms the binding domain of the enzyme. The values of torsion angles (phi, psi, chi(1) and chi(2)) for both the backbone as well as for the side-chain of Trp31 in molecules A and B are -94 degrees, -30 degrees, -66 degrees, 116 degrees and -128 degrees, 170 degrees, -63 degrees, -81 degrees, respectively. The conformation of Trp31 in molecule A is suitable for binding, while that in B hinders the passage of the ligand to the binding site. Consequently, alpha-tocopherol is able to bind to molecule A only, while the binding site of molecule B contains three water molecules. In the complex, the aromatic moiety of alpha-tocopherol is placed in the large space at the active site of the enzyme, while the long hydrophobic channel in the enzyme is filled by hydrocarbon chain of alpha-tocopherol. The critical interactions between the enzyme and alpha-tocopherol are generated between the hydroxyl group of the six-membered ring of alpha-tocopherol and His48 N(delta1) and Asp49 O(delta1) as characteristic hydrogen bonds. The remaining part of alpha-tocopherol interacts extensively with the residues of the hydrophobic channel of the enzyme, giving rise to a number of hydrophobic interactions, resulting in the formation of a stable complex.

Structure and function of proteins involved in milk allergies.

Allergy to milk proteins has been defined as any adverse reaction mediated by immunological mechanisms to one or several of proteins found in milk. The milk allergy has been classified according to the onset of symptoms as immediate or delayed type. The milk allergy seems to be manifested by three major proteins found in milk: alpha-lactalbumin, beta-lactoglobulin and caseins. The structural comparison of allergenic sites in alpha-lactalbumin and beta-lactoglobulin with the structure of lactoferrin has clearly shown that yet another major milk protein lactoferrin also possesses allergenic sites and thus may qualify to be an allergen. The heat treatment of milk proteins considerably reduces their allergenicity.

Isolation and characterization of novel protein with anti-fungal and anti-inflammatory properties from Aloe vera leaf gel.

The Aloe protein of 14 kDa from the Aloe vera leaf gel was isolated by an ion exchange chromatography using DEAE-cellulose and CM-cellulose column. The purified Aloe protein exhibited a potent anti-fungal activity against Candida paraprilosis, Candida krusei and Candida albicans. In addition, the purified Aloe protein also showed an anti-inflammatory property against pure lipoxygenase and cyclooxygenase-2 with 84% and 73% inhibition, respectively, and was verified by binding with these proteins by real time method by the phenomenon of surface plasmon resonance. This Aloe protein is a novel protein possessing antifungal and anti-inflammatory properties and thus sets a platform to be used as a medicinal plant product.

Proteomic analysis of heparin-binding proteins from human seminal plasma: a step towards identification of molecular markers of male fertility.

Glycosaminoglycans, especially heparin, are involved in various cell processes such as apoptosis, cell cycle control, platelet activation, capacitation, acrosome reaction and sperm decondensation. Heparin-binding proteins (HBPs) are essential constituents of human seminal fluid, which bind to sperm lipids containing the phosphorylcholine group and mediate the fertilization process. We utilized a proteomic set-up consisting of affinity chromatography, isoelectric focusing (IEF) coupled with matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry (MALDI TOF/MS) for protein analysis of human HBPs. We resolved 70 different spots on two-dimensional (2-D) gel and subsequently identified these proteins. Forty different types of proteins were identified. Functional analysis revealed that 38% of the proteins belonged to the enzyme category, 20% were involved in RNA processing and transcription, 18% in structure and transport function, and 16% in cell recognition and signal transduction. We also identified 8% of proteins with unknown functions, although their expression in seminal fluid has been documented. Proteins of seminal fluid that bind heparin may be directly involved in sperm capacitation and acrosome reaction (AR), which are the two critical steps for fertilization. This information on HBPs would be useful for identifying potential biomarkers of fertility in the near future.

Prolactin inducible protein in cancer, fertility and immunoregulation: structure, function and its clinical implications

Abstract.Prolactin inducible protein (PIP) is a 17- kDa single polypeptide chain, known by various names due to its versatile nature and function in human reproductive and immunological systems. It is expressed in several exocrine tissues such as the lacrimal, salivary, and sweat glands. Its expression is up regulated by prolactin and androgens, and estrogens down regulate it. Due to its over-expression in metastatic breast and prostate cancer, presently PIP is considered as a prognostic biomarker. Moreover, its aspartyl-proteinase nature suggests its role in tumor progression. PIP has unique features because it is small in size and plays multiple important functions. Its ability to bind potentially with CD4-T cell receptor, immunoglobulin G (IgG), actin, zinc α2-glycoprotein (ZAG), fibronectin and enamel pellicle, reveals its important biological functions. This is the first comprehensive review on the structure and functional analysis of PIP and its clinical applications.

Crystal structure of the novel complex formed between zinc alpha2-glycoprotein (ZAG) and prolactin-inducible protein (PIP) from human seminal plasma

This is the first report on the formation of a complex between zinc alpha2-glycoprotein (ZAG) and prolactin-inducible protein (PIP). The complex was purified from human seminal plasma and crystallized using 20% polyethylene glycol 9000 and 5% hexaethylene glycol. The structure of the complex has been determined using X-ray crystallographic method and refined to an R(cryst) of 0.199 (R(free)=0.239). The structure of ZAG is broadly similar to the structure of serum ZAG. The scaffolding of PIP consists of seven beta-strands that are organized in the form of two antiparallel beta-pleated sheets, resulting in the formation of a sandwiched beta-sheet. The amino acid sequence of PIP contains one potential N-glycosylation site at Asn77, and the same is found glycosylated with four sugar residues. The structure of the complex shows that the beta-structure of PIP is ideally aligned with the beta-structure of domain alpha3 of ZAG to form a long interface between two proteins. The proximal beta-strands at the long interface are arranged in an antiparallel manner. There are 12 hydrogen bonds and three salt bridges between ZAG and PIP. At the two ends of vertical interface, two salt bridges are formed between pairs of Lys41-Asp233 and Lys68-Glu229. On the perpendicular interface involving alpha1-alpha2 domains of ZAG and a loop of PIP, another salt bridge is formed. The internal space at the corner of the L-shaped structure is filled with solvent molecules including a carbonate ion. The overall buried area in the complex is approximately 914 A(2), which is considerably higher than the 660 A(2) reported for the class I major histocompatibility complex structures.

Current Overview of Allergens of Plant Pathogenesis Related Protein Families

Pathogenesis related (PR) proteins are one of the major sources of plant derived allergens. These proteins are induced by the plants as a defense response system in stress conditions like microbial and insect infections, wounding, exposure to harsh chemicals, and atmospheric conditions. However, some plant tissues that are more exposed to environmental conditions like UV irradiation and insect or fungal attacks express these proteins constitutively. These proteins are mostly resistant to proteases and most of them show considerable stability at low pH. Many of these plant pathogenesis related proteins are found to act as food allergens, latex allergens, and pollen allergens. Proteins having similar amino acid sequences among the members of PR proteins may be responsible for cross-reactivity among allergens from diverse plants. This review analyzes the different pathogenesis related protein families that have been reported as allergens. Proteins of these families have been characterized in regard to their biological functions, amino acid sequence, and cross-reactivity. The three-dimensional structures of some of these allergens have also been evaluated to elucidate the antigenic determinants of these molecules and to explain the cross-reactivity among the various allergens.