Isolation and characterization of a cDNA coding for human myeloperoxidase

Area of Science:

• Molecular Biology
• Biochemistry
• Cell Biology

Background:

• Myeloperoxidase (MPO) is a key enzyme in the innate immune system, crucial for host defense against microbial pathogens.
• Understanding the synthesis and processing of MPO is essential for comprehending its role in inflammatory diseases and developing targeted therapies.

Purpose of the Study:

• To isolate and characterize a cDNA encoding the carboxyl-terminal fragment of the human myeloperoxidase heavy chain.
• To determine the precise locations of the myeloperoxidase light and heavy chains within the polypeptide precursor.
• To elucidate the synthesis and processing pathway of human myeloperoxidase.

Main Methods:

• Construction and screening of a cDNA library from human leukemia HL-60 cells using differential and oligonucleotide hybridization.
• RNA transfer blot analysis to determine myeloperoxidase mRNA size.
• In vitro translation and immunoprecipitation assays to identify the MPO precursor polypeptide.
• Affinity chromatography to isolate heavy and light chain-specific antibodies.

Main Results:

• A cDNA clone encoding a 158-residue amino acid sequence, including a known heavy chain segment, was isolated.
• RNA transfer blot analysis revealed an MPO mRNA size of approximately 3.3 kb.
• In vitro translation produced a 74,000-Da precursor polypeptide that reacted with anti-MPO antibodies.
• Specific antibodies confirmed that the 74,000-Da precursor is processed into both light and heavy chains, with the heavy chain derived from the carboxyl terminus.

Conclusions:

• Human myeloperoxidase is synthesized as a single polypeptide precursor.
• This precursor undergoes post-translational processing to yield the mature light and heavy chains.
• The heavy chain of myeloperoxidase originates from the carboxyl-terminal portion of the precursor polypeptide.