PS 07-20 STRUCTURAL ANALYSIS OF GLYCOSYL CHAIN AT 14TH AMINO ACID OF ANGIOTENSINOGEN IN HUMAN PLASMA

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Abstract

Objective:

The Renin angiotensin system (RAS) is a major regulator of body fluid balance and control of blood pressure. The protease enzyme renin secreted from kidneys cleaves specifically angiotensinogen (Aogen) circulating in the blood to produce angiotensin I (Ang I). Human Aogen is a heterogeneous glycoprotein constitutively secreted by the liver. In addition, human Aogen contains four putative asparagine-linked glycosylation sites (Asn 14, 137, 271, 295) and contains four cysteines (Cys 18, 138, 232, 308), with Cys18 and Cys138 linked by a disulfide bridge. The glycosyl chains and cysteines position are very important for binding of the renin.

Objective:

Big angiotensin-25 (Bang-25) is a consisting of 25 amino acids with glycosyl chain (14th amino asid) and added cysteine (18th amino acid), which we recently isolated from human urine. To compare glycosyl chain of Bang-25 and Aogen, we analyzed of structure glycosyl chain at position 14th amino acid of human Aogen in plasma.

Design and Method:

To determine of glycosyl chain at position 14th amino acid, we performed lysyl endopeptidase digestion and reduction on human plasma Aogen. Then, Aogen after digest was purified by reverse-phase high-performance liquid chromatograpy (RP-HPLC), and glycosyl chain structure analyzed by the two-/three-dimensional HPLC mapping method.

Results:

We show that plasma Aogen has three types of glycosyl chain at position 14th amino acid. One glycosyl chain structure is identical to Bang-25 in urine. N-linked glycosylation on 14th amino acid of Aogen plays an important role about renin reaction. In addition, Bang-25 is rapidly cleaved by chymase to Ang II, but is resistant to cleave by renin.

Conclusions:

These results suggest that the structure of the glycosyl chain at position 14th amino acid of the human Aogen may be involved in the substrate specificity for renin or chymase.

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