Cleavage of proteoglycan aggregate by leucocyte elastase

Area of Science:

• Biochemistry
• Proteomics
• Glycobiology

Background:

• Aggrecan is a major proteoglycan in cartilage, crucial for tissue hydration and mechanical properties.
• Human leucocyte elastase (HLE) is a serine protease implicated in tissue degradation, including cartilage breakdown in inflammatory conditions.

Purpose of the Study:

• To investigate the specific cleavage sites and resulting fragments of aggrecan core protein upon digestion with human leucocyte elastase.
• To characterize the glycosaminoglycan stubs and N-terminal sequences of these degradation products.

Main Methods:

• Partial and complete digestion of proteoglycan aggregate with human leucocyte elastase.
• Analysis of degradation products using SDS-polyacrylamide electrophoresis (SDS-PAGE).
• Characterization of resulting bands by N-terminal amino acid sequencing and identification of attached glycosaminoglycan stubs (chondroitin and keratan sulfate).

Main Results:

• HLE digestion yielded aggrecan fragments of Mr 190,000, 140,000, 88,000, and 71,000 Da.
• Larger fragments (190,000 and 140,000 Da) contained chondroitin and keratan sulfate, with N-terminal sequences matching residues from position 398 of aggrecan core protein.
• Smaller fragments (88,000 and 71,000 Da) contained only keratan sulfate stubs.
• Complete digestion produced 71,000 Da (globular domain G2) and 56,000 Da (globular domain G1) fragments.

Conclusions:

• HLE cleaves the aggrecan core protein at the Val397-Ile398 bond within the interglobular domain linking G1 and G2.
• Further HLE digestion can occur within the chondroitin sulfate attachment domains of aggrecan.