Abstract
Information regarding molecular characteristics of leptin protein in different animal species in-cluding fish is scarce. With the aim of characterizing the native leptin protein of Indian major carprohu (Labeo rohita), at molecular level, the present study was designed to isolate rohu leptin from its hepatocytes (the prime source of leptin in fish) and immunobiochemical characterization of the same, subsequently. In the present study, chemical treatment and ultra-sonication technique was used for isolating leptin from rohu liver tissue. Purification of the protein was attempted using affinity column chromatography. The molecular, biophysical and serological characterization of rohu leptin was carried out by 2D-gel electrophoreis, SDS-PAGE, MALDI-TOF Mass spectroscopy and Western blot. The SDS-PAGE and 2D gel analysis revealed that rohu native leptin possesses molecular mass of 16 kDa. Western blot analysis showed that the fish hepatocytes possessed the sero-reactive leptin protein of 16 kDa. MALDI-TOF mass spectroscopy and peptide analysis showed the molecular mass of rohu leptin as 16283.38 Da. The serodiagnostic potential of native leptin of rohu was revealed for the first time while assessing its serological responses by ELISA using anti-leptin antibodies.
Highlights
Leptin is a 16 kDa adipocyte-derived cytokine like protein hormone composed of 167 amino acid residues produced mainly by adipose tissue and, at a lower extent, by other organs such as stomach, placenta, muscle, immune cells and mammary gland [1]
The present communication reveals for the first time the sero-diagnostic potential of fish leptin that might be exploited while preparing of sero-diagnostic tool(s) for fish leptin detection from unknown samples and/or measuring leptin concentration in blood, needed to select specific animal(s) for breeding purposes
The present work corroborates with the previous work of Cohen and co-workers where the molecular mass of affinity purified recombinant human leptin protein was estimated as approximately 16,000 Da by SDS-PAGE while the molecular mass of endogenous human leptin was reported as 16.026 ± 9 Da by MALDI mass technique [4]
Summary
Leptin is a 16 kDa adipocyte-derived cytokine like protein hormone composed of 167 amino acid residues produced mainly by adipose tissue and, at a lower extent, by other organs such as stomach, placenta, muscle, immune cells and mammary gland [1] It has been discovered in rodents where it is a synthesized primarily in adipose tissue and released into the blood stream [2]. Circulating concentrations of leptin may provide an indicator of fat content in live animals and facilitate more appropriate feeding and marketing strategies [5]. This important protein has not been studied and characterized in fish till date. The present communication reveals for the first time the sero-diagnostic potential of fish leptin that might be exploited while preparing of sero-diagnostic tool(s) for fish leptin detection from unknown (test) samples and/or measuring leptin concentration in blood, needed to select specific (individual) animal(s) for breeding purposes
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