Leucine-Rich Alpha-2-Glycoprotein NATIVE, Human Serum

Introduction to the Molecule

Leucine-rich alpha-2-glycoprotein (LRG) is a secretory type 1 acute phase protein whose expression is upregulated by the mediators of acute-phase response. The level of LRG in the blood increases during microbial infections and cancer, presumably as a result of the liver’s response to pro-inflammatory cytokines . The function of LRG is unknown but it is proved that LRG is tightly binding to Cyt C. It is suggested that extracellular Cyt C that is released from the cell death by an acute inflammation could be trapped to form tight complex with LRG and be excluded from circulation. LRG secretion by human hepatoma cells is increased by treatment with a mixture of cytokines including IL-6, IL-1b, and TNFα that are known to induce acute-phase proteins. It is known that LRG plays a role in adhesive interactions between lymphocytes and the endothelium. LRG may aid in the control of granulopoiesis by helping to modulate the surface expression of different receptor types, including transforming growth factor receptor, granulocyte-macrophage colony-stimulating factor receptor and possibly the macrophage colony stimulating factor receptor. LRG normal plasma concentration is approximately of 21 µg/ml based on purification, or ~50 µg/ml measured by ELISA. Human LRG is a serum glycoprotein of 312 amino acids in length with a predicted unmodified molecular weight of 34 to 36 kDa. SDS PAGE results show LRG molecular weight ranges from 44 to 55 kDa with isoelectric points ranging from 4.52 to 4.72, suggesting that 3 of 30 modifications occur. Leucine-rich alpha-2-glycoprotein consists of a single polypeptide chain with one galactosamine and four glucosamine oligosaccharides attached. The polypeptide has two intrachain disulfide bonds and contains 312 amino acid residues of which 66 are leucine. The amino acid sequence can be exactly divided into 13 segments of 24 residues each, eight of which exhibit a periodic pattern in the occurrence of leucine, proline, and asparagine.

Research topic

Sepsis

Description

Total 312 AA. MW: 34,35 kDa (calculated without glycosylation)

Amino Acid Sequence

Source

Human pooled serum (All blood samples used for protein preparation were tested and found negative for HBsAg, anti-HCV, HIV Ag/Ab, and syphilis.)

SDS-PAGE gel

SDS-PAGE analysis of human Leucine-rich alpha-2-glycoprotein-1 native protein, 12% gel stained with Coomassie Brillant Blue R250 1. M.W. marker – 14, 21, 31, 45, 66, 97 kDa 2. reduced and heated sample, 2.5μg/lane 3. non-reduced and non-heated sample, 2.5μg/lane

Biological Activity

Protein specifically bind to human Cytochrome c1, heme protein – tested.

Endotoxin

< 1.0 EU/ug

Formulation

Each vial contains 0.05 mg of protein lyophilized from 20mM TRIS, 20mM NaCl, pH 8.0

Reconstitution

Add deionized water to prepare a working stock solution of approximately 0.5 mg/mL and let the lyophilized pellet dissolve completely. Product is not sterile! Please filter the product by an appropriate sterile filter before using it in the cell culture.

Storage, Stability/Shelf Life

Store lyophilized protein at –20°C. Lyophilized protein remains stable until the expiry date when stored at –20°C. Aliquot reconstituted protein to avoid repeated freezing/thawing cycles and store at –80°C for long term storage. Reconstituted protein can be stored at 4°C for a limited period of time; it does not show any change after one week at 4°C.

Quality Control Test

BCA to determine quantity of the protein.

SDS PAGE to determine purity of the protein.

Gel permeation chromatography to determine purity and oligomeric state of the native protein.

LAL to determine quantity of endotoxin.

Identification of LRG from human serum by LC-MS/MS – The band of related protein was excised from the polyacrylamide gel and in-gel digested by trypsin (20ng/µL) for 2 hours in 40°C. Tryptic peptides were analysed by LC-MS/MS. The MASCOT score for LRG was 569 with 28% sequence coverage. MS/MS analysis of seven peptides yielded significant individual ions scores (for scores >50, p<0.05)

Applications

Cell culture and/or animal studies, ELISA, Immunological methods, Western blotting

References

• Ai J, Druhan LJ, Hunter MG, Loveland MJ, Avalos BR. LRG-accelerated differentiation defines unique G-CSFR signaling pathways downstream of PU.1 and C/EBPepsilon that modulate neutrophil activation. J Leukoc Biol. 2008 May;83 (5):1277-85
• Andersen JD, Boylan KL, Jemmerson R, Geller MA, Misemer B, Harrington KM, Weivoda S, Witthuhn BA, Argenta P, Vogel RI, Skubitz AP. Leucine-rich alpha-2-glycoprotein-1 is upregulated in sera and tumors of ovarian cancer patients. J Ovarian Res. 2010;3:21
• Appel RD, Hoogland C, Mostaguir K, Sanchez JC, Hochstrasser DF. SWISS-2DPAGE, ten years later. Proteomics. 2004 Aug;4 (8):2352-6
• Codina R, Vanasse A, Kelekar A, Vezys V, Jemmerson R. Cytochrome c-induced lymphocyte death from the outside in: inhibition by serum leucine-rich alpha-2-glycoprotein-1. Apoptosis. 2010 Feb;15 (2):139-52
• Haupt H, Baudner S. [Isolation and characterization of an unknown, leucine-rich 3.1-S-alpha2-glycoprotein from human serum (author's transl)]. Hoppe Seylers Z Physiol Chem. 1977 Jun;358 (6):639-46
• Jemmerson R, Weivoda S, Andersen JD, Skogen A, Schlievert PM, Fontana D, Schacker T, Tuite P, Dubinsky JM. ELISA for human serum leucine-rich alpha-2-glycoprotein-1 employing cytochrome c as the capturing ligand. J Immunol Methods. 2008 Jul 20;336 (1):22-9

By research topic:

• Sepsis

By molecule:

• Leucine-Rich Alpha-2-Glycoprotein

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