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Purification and characterization of a mitogenic lectin from cephalosporium, a pathogenic fungus causing mycotic keratitis.

Nagre NN, Chachadi VB, Eligar SM, Shubhada C, Pujari R, Shastry P, Swamy BM, Inamdar SR - Biochem Res Int (2010)

Bottom Line: Ophthalmic mycoses caused by infectious fungi are being recognized as a serious concern since they lead to total blindness.CSL showed strong binding to normal human peripheral blood mononuclear cells (PBMCs) to elicit mitogenic activity.The sugar specificity of the lectin and its interaction with PBMCs to exhibit mitogenic effect indicate its possible role in adhesion and infection process of Cephalosporium.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Karnatak University, Dharwad 580 003, Karnataka, India.

ABSTRACT
Ophthalmic mycoses caused by infectious fungi are being recognized as a serious concern since they lead to total blindness. Cephalosporium is one amongst several opportunistic fungal species implicated in ophthalmic infections leading to mycotic keratitis. A mitogenic lectin has been purified from the mycelia of fungus Cephalosporium, isolated from the corneal smears of a keratitis patient. Cephalosporium lectin (CSL) is a tetramer with subunit mass of 14 kDa, agglutinates human A, B, and O erythrocytes, and exhibits high affinity for mucin compared to fetuin and asialofetuin but does not bind to simple sugars indicating its complex sugar specificity. CSL showed strong binding to normal human peripheral blood mononuclear cells (PBMCs) to elicit mitogenic activity. The sugar specificity of the lectin and its interaction with PBMCs to exhibit mitogenic effect indicate its possible role in adhesion and infection process of Cephalosporium.

No MeSH data available.


Related in: MedlinePlus

Calibration curve for the estimation of molecular weight of CSL by gel filtration chromatography. X-axis represents the log molecular weight and Y-axis represents K average, marker proteins; BSA (66 kDa), ovalbumin (45 kDa), pepsin (34.7 kDa), chymotrypsinogen (25.6 kDa) and lysozyme (14.3 kDa).
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fig2: Calibration curve for the estimation of molecular weight of CSL by gel filtration chromatography. X-axis represents the log molecular weight and Y-axis represents K average, marker proteins; BSA (66 kDa), ovalbumin (45 kDa), pepsin (34.7 kDa), chymotrypsinogen (25.6 kDa) and lysozyme (14.3 kDa).

Mentions: The lectin was purified to homogeneity in a single step by affinity chromatography on asialofetuin-Sepharose 4B column (Figure 1). The fold purification and the total % recovery of the purified lectin from 1 g of the dry mycelial powder are summarized in Table 1; the minimum concentration of the protein required for agglutination (MCA) was found to be 1.145 μg for the crude extract and 0.021 μg for the purified lectin. The eluted lectin was found to be homogenous as revealed by single band on SDS-PAGE in 15% gel (Figure 1, Inset). Subunit molecular mass of 14 kDa was estimated for the lectin by SDS-PAGE, whereas molecular mass of 57 kDa was estimated by gel filtration chromatography (Figure 2), suggesting tetrameric nature of the lectin. Purified CSL agglutinated the human erythrocytes of all blood groups, indicating that it has a blood group nonspecific nature. Hapten inhibition studies showed that the hemagglutinating activity of CSL was inhibited by mucin, fetuin, and asialofetuin, with mucin being the most potent inhibitor with minimum inhibitory concentration (MIC) of 0.785 μg/50 μl (Table 2).


Purification and characterization of a mitogenic lectin from cephalosporium, a pathogenic fungus causing mycotic keratitis.

Nagre NN, Chachadi VB, Eligar SM, Shubhada C, Pujari R, Shastry P, Swamy BM, Inamdar SR - Biochem Res Int (2010)

Calibration curve for the estimation of molecular weight of CSL by gel filtration chromatography. X-axis represents the log molecular weight and Y-axis represents K average, marker proteins; BSA (66 kDa), ovalbumin (45 kDa), pepsin (34.7 kDa), chymotrypsinogen (25.6 kDa) and lysozyme (14.3 kDa).
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3008968&req=5

fig2: Calibration curve for the estimation of molecular weight of CSL by gel filtration chromatography. X-axis represents the log molecular weight and Y-axis represents K average, marker proteins; BSA (66 kDa), ovalbumin (45 kDa), pepsin (34.7 kDa), chymotrypsinogen (25.6 kDa) and lysozyme (14.3 kDa).
Mentions: The lectin was purified to homogeneity in a single step by affinity chromatography on asialofetuin-Sepharose 4B column (Figure 1). The fold purification and the total % recovery of the purified lectin from 1 g of the dry mycelial powder are summarized in Table 1; the minimum concentration of the protein required for agglutination (MCA) was found to be 1.145 μg for the crude extract and 0.021 μg for the purified lectin. The eluted lectin was found to be homogenous as revealed by single band on SDS-PAGE in 15% gel (Figure 1, Inset). Subunit molecular mass of 14 kDa was estimated for the lectin by SDS-PAGE, whereas molecular mass of 57 kDa was estimated by gel filtration chromatography (Figure 2), suggesting tetrameric nature of the lectin. Purified CSL agglutinated the human erythrocytes of all blood groups, indicating that it has a blood group nonspecific nature. Hapten inhibition studies showed that the hemagglutinating activity of CSL was inhibited by mucin, fetuin, and asialofetuin, with mucin being the most potent inhibitor with minimum inhibitory concentration (MIC) of 0.785 μg/50 μl (Table 2).

Bottom Line: Ophthalmic mycoses caused by infectious fungi are being recognized as a serious concern since they lead to total blindness.CSL showed strong binding to normal human peripheral blood mononuclear cells (PBMCs) to elicit mitogenic activity.The sugar specificity of the lectin and its interaction with PBMCs to exhibit mitogenic effect indicate its possible role in adhesion and infection process of Cephalosporium.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Karnatak University, Dharwad 580 003, Karnataka, India.

ABSTRACT
Ophthalmic mycoses caused by infectious fungi are being recognized as a serious concern since they lead to total blindness. Cephalosporium is one amongst several opportunistic fungal species implicated in ophthalmic infections leading to mycotic keratitis. A mitogenic lectin has been purified from the mycelia of fungus Cephalosporium, isolated from the corneal smears of a keratitis patient. Cephalosporium lectin (CSL) is a tetramer with subunit mass of 14 kDa, agglutinates human A, B, and O erythrocytes, and exhibits high affinity for mucin compared to fetuin and asialofetuin but does not bind to simple sugars indicating its complex sugar specificity. CSL showed strong binding to normal human peripheral blood mononuclear cells (PBMCs) to elicit mitogenic activity. The sugar specificity of the lectin and its interaction with PBMCs to exhibit mitogenic effect indicate its possible role in adhesion and infection process of Cephalosporium.

No MeSH data available.


Related in: MedlinePlus