Shedding of cell membrane-bound proteoglycans

Abstract

Membrane-bound proteoglycans function primarily as coreceptors for many glycosaminoglycan (GAG)-binding ligands at the cell surface. The majority of membrane-bound proteoglycans can also function as soluble autocrine or paracrine effectors as their extracellular domains, replete with all GAG chains, are enzymatically cleaved and released from the cell surface by ectodomain shedding. In particular, the ectodomain shedding of syndecans, a major family of cell surface heparan sulfate proteoglycans, is an important posttranslational mechanism that modulates diverse pathophysiological processes. Syndecan shedding is a tightly controlled process that regulates the onset, progression, and resolution of various infectious and noninfectious inflammatory diseases. This review describes methods to induce and measure the shedding of cell membrane-bound proteoglycans, focusing on syndecan shedding as a prototypic example.

Fig. 1

Time-dependent induction of syndecan-1 shedding by PMA measured by dot immunoblotting. Confluent normal murine mammary gland (NMuMG) cells in 96-well plates were incubated with 1 µM PMA for 0, 15, or 30 min at 37°C in triplicate. For quantification of shed syndecan-1 ectodomains, 35 µL of the conditioned medium was dot blotted onto the Immobilon Ny+ membrane. For quantification of cell surface syndecan-1, following removal of the conditioned medium, cells were washed once with ice-cold TBS/EDTA and incubated with 50 µL of ice-cold 10 µg/mL TPCK-treated trypsin for 10 min at 4°C. Trypsin was inactivated with 50 µL of 100 µg/mL soybean trypsin inhibitor. Trypsinates (35 µL) were dot blotted onto the Immobilon Ny+ membrane. The membrane was immunoblotted with 281–2 antibodies, developed, and quantified as described. (a) Picture of confluent NMuMG cells. (b) Developed dot blot. (c) Standard curve of syndecan-1. (d) Quantification of shed and cell surface syndecan-1 at various times post-PMA stimulation.

Fig. 2

Time-dependent reduction of cell surface syndecan-1 and -4 by PMA measured by FACS. Confluent NMuMG cells in 6-well plates were incubated with 1 µM PMA for 15 or 30 min at 37°C. Cells were detached by incubating with PBS/EDTA and treated with primary antibodies (281–2 for syndecan-1; Ky8.2 for syndecan-4) and Alexa 488 anti-rat secondary antibodies. After fixing cells with 2% paraformaldehyde (PFA), the fluorescence associated with cell surfaces was measured by FACS. Black line, cell surface syndecans without PMA treatment; light gray line, cell surface syndecans 15 min after PMA treatment; dark gray line, cell surface syndecans 30 min after PMA treatment; and dotted line, background fluorescence measured with secondary antibody only.