The synthesis, characterization, and use of a protein-cysteine proteinase inhibitor complex for the study of endosome/lysosome fusion

The cysteine proteinases cathepsins B, L, and S are lysosomal enzymes responsible for the degradation of endocytosed proteins. Their presence in human cell monocytic lines THP1 and U937 was detected by the use of the membrane-permeable, irreversible, active-site directed inhibitor Fmoc-(¹²⁵I)Tyr- Ala-CHN₂ followed by immunoprecipitation of the enzymes, SDSPAGE, and autoradiography. All three enzymes were detected in THP1 cells; only after differentiation of U937 cells to macrophage-like cells were the enzymes detectable. Both cell lines show multiple forms of cathepsin S, at 35 kDa, 28 kDa, and 26 kDa, suggesting the presence of an active pro-form of cathepsin S as well as the processing of cathepsin S into single- and two-chain forms. This is the first evidence for an active pro-form of a cysteine proteinase and for the processing of cathepsin S to a two-chain enzyme form. Multiple forms of cathepsin L were analyzed by isoelectric focusing followed by denaturing polyacrylamide gel electrophoresis. The multiple forms are not due to the presence of carbohydrate chains on the protein. The inhibitor Fmoc-Tyr-Ala-CHN₂ synthesized and its inhibitory properties against cathepsins B, L, and S were determined. Both in vitro and in vivo studies show that this inhibitor is an effective reagent for studying lysosomal cysteine proteinases. In order to be useful in the study of the delivery of lysosomal enzymes to vesicles containing recently internalized compounds, the deblocked peptidyl diazomethane inhibitor NH₂-Tyr-Ala-CHN₂ was cross-linked to bovine serum albumin (BSA) using the heterobifunctional crosslinking agent sulfo-SANPAH. This non-reducible cross-linked complex was used to characterize the inhibitory properties of the protein-inhibitor complex against cathepsins B, L, S and papain in vitro and in vivo.

Name thesis:degree_name

Ph. D.

Level thesis:degree_level

doctoral

Discipline thesis:degree_discipline

Biochemistry and Anaerobic Microbiology

Department dc:contributor.department

Biochemistry and Anaerobic Microbiology

Grantor dc:publisher

Virginia Tech

Year dc:date.issued

1994

Author dc:creator

• Mountz, Adele K.

Chair dc:contributor.committeechair

• Mason, R.W.

Committee members dc:contributor.committeemember

• Newton, William E.
• Castagnoli, Neal Jr.
• Gregory, Eugene "Mick"
• Gregory, D.R.

Statement dc:rights

• In Copyright

Licence dc:rights.uri

• http://rightsstatements.org/vocab/InC/1.0/

Language dc:language.iso

en

Dc Identifier Other

etd-06072006-124205

OAI identifier oai:identifier

oai:vtechworks.lib.vt.edu:10919/38555