Cloning and characterization of complementary DNA for human tryptase

Area of Science:

• Biochemistry
• Molecular Biology
• Immunology

Background:

• Human mast cell tryptase is a major neutral protease and a specific marker for mast cells.
• It indicates mast cell activation when found in biological fluids.
• Understanding its structure is crucial for its role in cellular processes.

Purpose of the Study:

• To determine the complete amino acid sequence of human mast cell tryptase.
• To identify structural features, including catalytic sites and potential glycosylation sites.
• To compare the human sequence with other serine proteases, particularly dog tryptase.

Main Methods:

• Complementary DNA (cDNA) cloning from a lambda ZAP library.
• mRNA isolation from human mast cell preparations.
• Nucleic acid sequencing to deduce the amino acid sequence.

Main Results:

• The human tryptase sequence comprises a 244-amino acid catalytic portion and a 30-amino acid leader sequence.
• Two N-linked carbohydrate binding sites and a catalytic triad (His74, Asp120, Ser223) were identified.
• High sequence similarity was found with dog tryptase (84% catalytic, 67% leader).
• Specific amino acid residues (Asp217, Glu245, Asp244) suggest specificity for basic residues (Arg, Lys) in substrates.

Conclusions:

• The determined amino acid sequence provides a foundation for understanding human mast cell tryptase structure and function.
• Specific residues within the substrate binding pocket suggest a preference for basic amino acids, influencing substrate cleavage.
• Further research can now explore structure-function relationships in detail.