Krishnan Hajela

Cleavage of Kininogen and Subsequent Bradykinin Release by the Complement Component: Mannose-Binding Lectin-Associated Serine Protease (MASP)-1

Bradykinin (BK), generated from high-molecular-weight kininogen (HK) is the major mediator of swelling attacks in hereditary angioedema (HAE), a disease associated with C1-inhibitor deficiency. Plasma kallikrein, activated by factor XIIa, is responsible for most of HK cleavage. However other proteases, which activate during episodes of angioedema, might also contribute to BK production. The lectin pathway of the complement system activates after infection and oxidative stress on endothelial cells generating active serine proteases: MASP-1 and MASP-2. Our aim was to study whether activated MASPs are able to digest HK to release BK. Initially we were trying to find potential new substrates of MASP-1 in human plasma by differential gel electrophoresis, and we identified kininogen cleavage products by this proteomic approach. As a control, MASP-2 was included in the study in addition to MASP-1 and kallikrein. The proteolytic cleavage of HK by MASPs was followed by SDS-PAGE, and BK release was detected by HPLC. We showed that MASP-1 was able to cleave HK resulting in BK production. MASP-2 could also cleave HK but could not release BK. The cleavage pattern of MASPs is similar but not strictly identical to that of kallikrein. The catalytic efficiency of HK cleavage by a recombinant version of MASP-1 and MASP-2 was about 4.0×102 and 2.7×102 M−1s−1, respectively. C1-inhibitor, the major inhibitor of factor XIIa and kallikrein, also prevented the cleavage of HK by MASPs. In all, a new factor XII- and kallikrein-independent mechanism of bradykinin production by MASP-1 was demonstrated, which may contribute to the pro-inflammatory effect of the lectin pathway of complement and to the elevated bradykinin levels in HAE patients.

ESR studies on the effect of ionic radii on displacement of Mn2+ bound to a soluble β-galactoside binding hepatic lectin

Binding of divalent metal ions to hepatic soluble β‐galactoside binding lectin was studied using electron spin resonance (ESR) spectroscopy. The Mn2+ bound to hepatic lectin could be displaced by Mg2+, Cu2+, Ni2+ and Ca2+ but not by Sr2+. As the ionic radii of Mg2+ (0.65 Å), Cu2+ (0.73 Å) and Ni2+ (0.72 Å) are appreciably smaller than Ca Mn2+ binding site is more accessible to Mg2+, Cu2+, and Ni2+ as compared to Ca2+, the ionic radius of Mn2+ being 0.80 Å. Sr2+ with an ionic radius of 1.13 is thus unable to displace bound Mn2+. Surprisingly, the presence of specific sugars like α‐lactose, or α‐d‐galactose facilitated the displacement of bound Mn2+ by metal ions whereas non‐specific sugars, i.e. α‐d‐glucose, β‐d‐fructose and α‐d‐ribose had no effect. It appears that minor perturbations in the saccharide binding site significantly affect the ability of the metal binding site to ligate bivalent metals.

CARBOHYDRATE INDUCED MODULATION OF CELL MEMBRANE. VII. BINDING OF EXOGENOUS LECTIN INCREASES OSMOFRAGILITY OF ERYTHROCYTES

Due to their multivalent binding character, lectins when added exogenously will cross‐link membrane surface receptors leading to lateral molecular reorganizations in the plane of the bilayer. This study reports for the first time that agglutination of rabbit erythrocytes by lentil lectin and concanavalin A increases their osmofragility. Increase in osmofragility was detected by measuring the hemolysis of erythrocytes in hypotonic as well as in isotonic solutions. It was also found that agglutination per se does not increase osmofragility but the binding of legume lectin is essential since human Rh+ cells agglutinated by a monoclonal antibody do not exhibit hemolysis.

Carbohydrate-induced modulation of cell membrane. VIII. Agglutination with mammalian lectin galectin-1 increases osmofragility and membrane fluidity of trypsinized erythrocytes

Interaction of lectins with cell surface determinants may alter membrane properties. Using trypsinized rabbit erythrocytes as model we tested the capacity of an endogenous lectin in this respect. Galectin‐1 is a member of an adhesion/growth‐regulatory family known to interact for example with ganglioside GM1 and also the hydrophobic tail of oncogenic H‐Ras. Assays on membrane fluidity and osmofragility detect galectin‐1s capacity to increase the parameters. Moreover, it increases susceptibility of erythrocytes to radical damage. These observations indicate the potential of this endogenous lectin to affect membrane properties beyond the immediate interaction with cell surface epitopes.

Oxidation of Goat Hepatic Galectin-1 Induces Change in Secondary Structure

Galectin-1 requires a reducing environment for its lectin activity and the carbohydrate binding function is destroyed in oxidizing condition. In this report we provide direct evidence that the oxidation of goat hepatic galectin-1 perturbs its carbohydrate recognition domain and this could be due to changes in secondary structure of goat hepatic galectin-1. Conformational changes in goat hepatic galectin-1 due to oxidation were investigated by absorption, fluorescence and circular dichroism measurements.

Carbohydrate induced modulation of cell membrane I. Interaction of sialic acid with peripheral blood lymphocytes: A spin label study

Sialic acid is now known to serve as ligand for lymphocyte lectins known as selectins. Although its role as a ligand for adhesion molecules has been studied extensively, no studies have been performed to determine the physiological changes in lymphocytes after binding of sialic acid to lymphocyte lectin. We report for the first time that interaction of lymphocytes with sialic acid severely restricts the rotational mobility of the cell surface proteins as well as membrane lipids, as studied by EPR spectroscopy using spin probes. Binding of mucin totally immobilizes the lymphocyte membrane. Surprisingly the binding of sialic acid or mucin also immobilized the aqueous probe TEMPO, indicating an appreciable increase in cytoplasmic viscosity.

Studies on a glucose-binding lectin from peripheral blood lymphocytes.

Lectin-carbohydrate interactions have been found to be important in many of the steps of lymphocyte recirculation and inflammatory responses. A D-glucose-specific lectin was isolated from goat peripheral blood lymphocytes by affinity chromatography on N-acetyl-D-glucosamine agarose and gave a single band corresponding to 112 kDa in SDS-PAGE, irrespective of treatment with 2-mercaptoethanol. The lymphocyte lectin agglutinated rabbit and human ABO erythrocytes, the hemagglutinating activity being Ca2+ dependent. It appears to be a member of type C animal lectins.

Mutational and expressional analyses of PTEN gene in colorectal cancer from Northern India

The PTEN is a tumor‐suppressor gene located on chromosome 10q23.3 and established to play key role in the varied types of cancer. To elucidate the possible effect of mutations and inactivation of PTEN gene on the occurrence and development of colorectal cancer (CRC), 223 cancer specimens were selected to probe PTEN gene mutations through the micro dissection of the genome. Polymerase chain reaction single‐strand conformation polymorphism and DNA sequencing methods were applied for mutations while protein expression was evaluated by immunohistochemistry. Mutations in exons 7 and 8 of PTEN were observed in 12.5% and PTEN loss of expression was identified in 48% in CRC. In exon 7, we found the insertion of “G” resulted into the change at codon 218 from TGC to GTC leading to change in the reading frame starting downward from Cystein to Valine. In addition, the insertion of “A” in the same exon at codon 213 resulted into the change of codon CCT to CCA, which cause silent mutation. In exon 8, however, “A” is replaced by C at codon 282, but both encodes for Glycine. Statistically significant loss of PTEN expression was observed in cancerous tissue when compared with the adjacent control (P < 0.05). Furthermore, weak PTEN expression in CRC tissues were significantly associated with tumor size, depth of invasion, lymphatic invasion, lymph node metastasis, grade of differentiation, and TNM stage (P < 0.05). Our results suggested that PTEN gene mutation and loss of PTEN expression may provide valuable prognostic information to aid treatment strategies for CRC patients.

Carbohydrate induced modulation of cell membrane. VI. Binding of exogenous lectin induces susceptibility of erythrocytes to free radical damage: a spin label study.

The oxidation of erythrocyte membrane has been widely used as a model to study the damage of biomembranes by free radicals. Whether binding of lectin to erythrocytes has any effect on peroxidant injury has never been studied. This study reports for the first time that crosslinking of erythrocyte surface glycoprotein by an exogenous lectin significantly enhances the susceptibility to membrane damage by free radicals, as evidenced by the increase in membrane fluidity measured by EPR using spin label and the increase in the amount of oxyhemoglobin liberated due to cell lysis.

Carbohydrate-induced modulation of cell membrane—III. Interaction of sialic acid and mannose with hamster splenic lymphocytes: A spin label study

The role of different carbohydrates as ligands for adhesion molecules has been extensively studied. However, the physiological changes in the splenic lymphocytes on binding these ligands have never been studied. In this paper, we report that binding of sialic acid or mannose to hamster splenic lymphocytes restricts the mobility of membrane proteins and lipids as studied by EPR spectroscopy using spin probes. Binding of mucin and heparin totally restricts the mobility probably due to crosslinking of the surface lectins. Binding of these ligands also results in an increase in the viscosity of the cytoplasm.