Extremely Stable Soluble High Molecular Mass Multi-Protein Complex with DNase Activity in Human Placental Tissue

Abstract

Human placenta is an organ which protects, feeds, and regulates the grooving of the embryo. Therefore, identification and characterization of placental components including proteins and their multi-protein complexes is an important step to understanding the placenta function. We have obtained and analyzed for the first time an extremely stable multi-protein complex (SPC, ∼1000 kDa) from the soluble fraction of three human placentas. By gel filtration on Sepharose-4B, the SPC was well separated from other proteins of the placenta extract. Light scattering measurements and gel filtration showed that the SPC is stable in the presence of NaCl, MgCl2, acetonitrile, guanidinium chloride, and Triton in high concentrations, but dissociates efficiently in the presence of 8 M urea, 50 mM EDTA, and 0.5 M NaCl. Such a stable complex is unlikely to be a casual associate of different proteins. According to SDS-PAGE and MALDI mass spectrometry data, this complex contains many major glycosylated proteins with low and moderate molecular masses (MMs) 4–14 kDa and several moderately abundant (79.3, 68.5, 52.8, and 27.2 kDa) as well as minor proteins with higher MMs. The SPC treatment with dithiothreitol led to a disappearance of some protein bands and revealed proteins with lower MMs. The SPCs from three placentas efficiently hydrolyzed plasmid supercoiled DNA with comparable rates and possess at least two DNA-binding sites with different affinities for a 12-mer oligonucleotide. Progress in study of placental protein complexes can promote understanding of their biological functions.

Extremely stable high molecular mass soluble multiprotein complex from eggs of sea urchin Strongylocentrotus intermedius with phosphatase activity

“…According to the LS data, the eggs SPCs were stable in 20mM Tris‐HCl, pH 7.5, containing 1M NaCl as well as 3M MgCl 2 (Figure 2A) destroying usually electrostatic contacts between different proteins. It was previously shown that 20mM Tris‐HCl, pH 7.5, containing 8M urea and 1.0M NaCl (buffer D) effectively destroyed very stable complexes from human milk and placentas . In buffer D, there was faster destroying of urchin SPCs leading to decrease in the LS (Figure 2A).…”

“…After dialysis, the homogenate was concentrated 10 times using a dialysis bag in an air stream at 4°C. Then, the homogenate (0.5‐1 mL) proteins were separated by FPLC gel filtration on a column with Sepharose 4B equilibrated with 20mM Tris‐HCl, pH 7.5, containing 0.15M NaCl similar to other studies; elution of proteins was carried out using the same buffer. All operations were performed using sterile conditions.…”

“…Seven hundred thirty‐three unique proteins as 34 known novel hetero‐oligomeric protein complexes were identified. Using different methods, we have recently analyzed a possibility of an existence of multiprotein complexes in the soluble fraction of a homogenate of human placenta and milk . The extremely stable multiprotein complexes (SPC; approximately 1000 ± 100 kDa) were separated from different placentas and milk .…”

“…It was shown, that these SPCs are stable in the presence of NaCl and MgCl 2 in high concentrations. The complexes dissociate only in the presence of 8M urea + 0.5M to 1.0M NaCl . According to SDS‐PAGE, the SPCs contain many but different proteins in the case of placentas and milk having molecular masses (MMs) from 14 to 79.3 kDa .…”

“…The complexes dissociate only in the presence of 8M urea + 0.5M to 1.0M NaCl . According to SDS‐PAGE, the SPCs contain many but different proteins in the case of placentas and milk having molecular masses (MMs) from 14 to 79.3 kDa . To our best of knowledge, no data concerning soluble protein complexes from an extract of sea urchin embryos have been reported.…”