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Opsonic function of sialic acid specific lectin in freshwater crab Paratelphusa jacquemontii.

Denis M, Thayappan K, Ramasamy SM, Munusamy A - Springerplus (2015)

Bottom Line: The flowcytometry observation affirmed the enhancement of phagocytosis by Pjlec coated hemocyte.The mechanism of phagocytosis and induction were dependent on specific recognition of the erythrocyte by the multivalent binding site of the lectin protein, and the elicitation of the immune response was a function of the sialoglycan surface.The pathway of the challenge suggested that after entry of nonself recognition by lectin was followed by induction and activation of phagocytosis by opsonic binding of the lectin.

View Article: PubMed Central - PubMed

Affiliation: Department of Zoology, University of Madras, Guindy Campus, Chennai, 600025 India.

ABSTRACT
The sialic acid specific humoral lectin, Pjlec of the freshwater crab Paratelphusa jacquemontii was investigated for its opsonin function with rabbit erythrocyte as target cell for phagocytosis by the crab's hemocyte. The untreated or trypsin treated erythrocyte induced lectin response after challenge however failed when treated with neuraminidase evidently indicating glycan dependency for elicited immune response. Our observation of in vitro phagocytosis of the erythrocyte untreated or coated with serum, clarified serum appeared to be recognized and engulfed by hemocytes but when coated with isolated lectin Pjlec, the response was elicited. Moreover, with trypsin treated erythrocyte the response remained unchanged but neuraminidase or O-glycosidase treatment eliminated the response reaction. This suggested the sialic acid specific reaction of lectin with the erythrocyte and was essential for recognition to allow the lectin Pjlec to act as an opsonin. The flowcytometry observation affirmed the enhancement of phagocytosis by Pjlec coated hemocyte. The efficiency of in vitro hemolysis of Pjlec coated erythrocyte with hemocyte when compared to untreated erythrocyte with or without hemocyte also established the opsonic function of the lectin. The mechanism of phagocytosis and induction were dependent on specific recognition of the erythrocyte by the multivalent binding site of the lectin protein, and the elicitation of the immune response was a function of the sialoglycan surface. The pathway of the challenge suggested that after entry of nonself recognition by lectin was followed by induction and activation of phagocytosis by opsonic binding of the lectin.

No MeSH data available.


Related in: MedlinePlus

Flow cytometry in vitro phagocytosis analysis of hemocyte population in the hemolymph of Paratelphusa jacquemontii. Flow cytogram of in vitro phagocytic activity observed in 100 µl hemocyte suspension in iso-osmotic buffer (Tris 50 mM, NaCl 210 mM, KCl 5 mM, MgCl2 2.5 mM, D-glucose 100 mg, pH 7.5, 480 mOsm) with glutaraldehyde fixed rabbit erythrocyte treated with enzymes and Pjlec. Mean SD: 94.6 2.1 (n 3). Scatter gram was generated by combining forward light scatter (FS) with 7-AAD fluorescence.a Untreated rabbit erythrocyte; b FITC labeled rabbit erythrocyte; c FITC labeled rabbit erythrocyte + hemocytes (2 min); d FITC labeled rabbit erythrocyte + hemocytes (30 min); e Lectin coated FITC labeled rabbit erythrocyte + hemocytes (2 min); f Pjlec lectin coated FITC labeled rabbit erythrocyte + hemocytes (30 min). g Trypsin treated FITC labeled rabbit erythrocyte + hemocytes (2 min); h Trypsin treated FITC labeled rabbit erythrocyte + hemocytes (30 min)
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Fig6: Flow cytometry in vitro phagocytosis analysis of hemocyte population in the hemolymph of Paratelphusa jacquemontii. Flow cytogram of in vitro phagocytic activity observed in 100 µl hemocyte suspension in iso-osmotic buffer (Tris 50 mM, NaCl 210 mM, KCl 5 mM, MgCl2 2.5 mM, D-glucose 100 mg, pH 7.5, 480 mOsm) with glutaraldehyde fixed rabbit erythrocyte treated with enzymes and Pjlec. Mean SD: 94.6 2.1 (n 3). Scatter gram was generated by combining forward light scatter (FS) with 7-AAD fluorescence.a Untreated rabbit erythrocyte; b FITC labeled rabbit erythrocyte; c FITC labeled rabbit erythrocyte + hemocytes (2 min); d FITC labeled rabbit erythrocyte + hemocytes (30 min); e Lectin coated FITC labeled rabbit erythrocyte + hemocytes (2 min); f Pjlec lectin coated FITC labeled rabbit erythrocyte + hemocytes (30 min). g Trypsin treated FITC labeled rabbit erythrocyte + hemocytes (2 min); h Trypsin treated FITC labeled rabbit erythrocyte + hemocytes (30 min)

Mentions: The flow cytometry analysis of in vivo phagocytosis by hemocytes of P. jacquemontii was undetected, however in vitro phagocytosis was observed up to 30 min with FITC labeled rabbit erythrocyte (Fig. 6a, b). The phagocytic activity of hemocytes against untreated rabbit erythrocyte was 33.74 % (Fig. 6c, d) and the trypsin treated erythrocyte 68.29 % (Fig. 6e, f), and with lectin coated rabbit erythrocyte 97 % (Fig. 6g, h).Fig. 6


Opsonic function of sialic acid specific lectin in freshwater crab Paratelphusa jacquemontii.

Denis M, Thayappan K, Ramasamy SM, Munusamy A - Springerplus (2015)

Flow cytometry in vitro phagocytosis analysis of hemocyte population in the hemolymph of Paratelphusa jacquemontii. Flow cytogram of in vitro phagocytic activity observed in 100 µl hemocyte suspension in iso-osmotic buffer (Tris 50 mM, NaCl 210 mM, KCl 5 mM, MgCl2 2.5 mM, D-glucose 100 mg, pH 7.5, 480 mOsm) with glutaraldehyde fixed rabbit erythrocyte treated with enzymes and Pjlec. Mean SD: 94.6 2.1 (n 3). Scatter gram was generated by combining forward light scatter (FS) with 7-AAD fluorescence.a Untreated rabbit erythrocyte; b FITC labeled rabbit erythrocyte; c FITC labeled rabbit erythrocyte + hemocytes (2 min); d FITC labeled rabbit erythrocyte + hemocytes (30 min); e Lectin coated FITC labeled rabbit erythrocyte + hemocytes (2 min); f Pjlec lectin coated FITC labeled rabbit erythrocyte + hemocytes (30 min). g Trypsin treated FITC labeled rabbit erythrocyte + hemocytes (2 min); h Trypsin treated FITC labeled rabbit erythrocyte + hemocytes (30 min)
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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Fig6: Flow cytometry in vitro phagocytosis analysis of hemocyte population in the hemolymph of Paratelphusa jacquemontii. Flow cytogram of in vitro phagocytic activity observed in 100 µl hemocyte suspension in iso-osmotic buffer (Tris 50 mM, NaCl 210 mM, KCl 5 mM, MgCl2 2.5 mM, D-glucose 100 mg, pH 7.5, 480 mOsm) with glutaraldehyde fixed rabbit erythrocyte treated with enzymes and Pjlec. Mean SD: 94.6 2.1 (n 3). Scatter gram was generated by combining forward light scatter (FS) with 7-AAD fluorescence.a Untreated rabbit erythrocyte; b FITC labeled rabbit erythrocyte; c FITC labeled rabbit erythrocyte + hemocytes (2 min); d FITC labeled rabbit erythrocyte + hemocytes (30 min); e Lectin coated FITC labeled rabbit erythrocyte + hemocytes (2 min); f Pjlec lectin coated FITC labeled rabbit erythrocyte + hemocytes (30 min). g Trypsin treated FITC labeled rabbit erythrocyte + hemocytes (2 min); h Trypsin treated FITC labeled rabbit erythrocyte + hemocytes (30 min)
Mentions: The flow cytometry analysis of in vivo phagocytosis by hemocytes of P. jacquemontii was undetected, however in vitro phagocytosis was observed up to 30 min with FITC labeled rabbit erythrocyte (Fig. 6a, b). The phagocytic activity of hemocytes against untreated rabbit erythrocyte was 33.74 % (Fig. 6c, d) and the trypsin treated erythrocyte 68.29 % (Fig. 6e, f), and with lectin coated rabbit erythrocyte 97 % (Fig. 6g, h).Fig. 6

Bottom Line: The flowcytometry observation affirmed the enhancement of phagocytosis by Pjlec coated hemocyte.The mechanism of phagocytosis and induction were dependent on specific recognition of the erythrocyte by the multivalent binding site of the lectin protein, and the elicitation of the immune response was a function of the sialoglycan surface.The pathway of the challenge suggested that after entry of nonself recognition by lectin was followed by induction and activation of phagocytosis by opsonic binding of the lectin.

View Article: PubMed Central - PubMed

Affiliation: Department of Zoology, University of Madras, Guindy Campus, Chennai, 600025 India.

ABSTRACT
The sialic acid specific humoral lectin, Pjlec of the freshwater crab Paratelphusa jacquemontii was investigated for its opsonin function with rabbit erythrocyte as target cell for phagocytosis by the crab's hemocyte. The untreated or trypsin treated erythrocyte induced lectin response after challenge however failed when treated with neuraminidase evidently indicating glycan dependency for elicited immune response. Our observation of in vitro phagocytosis of the erythrocyte untreated or coated with serum, clarified serum appeared to be recognized and engulfed by hemocytes but when coated with isolated lectin Pjlec, the response was elicited. Moreover, with trypsin treated erythrocyte the response remained unchanged but neuraminidase or O-glycosidase treatment eliminated the response reaction. This suggested the sialic acid specific reaction of lectin with the erythrocyte and was essential for recognition to allow the lectin Pjlec to act as an opsonin. The flowcytometry observation affirmed the enhancement of phagocytosis by Pjlec coated hemocyte. The efficiency of in vitro hemolysis of Pjlec coated erythrocyte with hemocyte when compared to untreated erythrocyte with or without hemocyte also established the opsonic function of the lectin. The mechanism of phagocytosis and induction were dependent on specific recognition of the erythrocyte by the multivalent binding site of the lectin protein, and the elicitation of the immune response was a function of the sialoglycan surface. The pathway of the challenge suggested that after entry of nonself recognition by lectin was followed by induction and activation of phagocytosis by opsonic binding of the lectin.

No MeSH data available.


Related in: MedlinePlus