Evidence that muscle cells do not express the histidine-rich glycoprotein associated with AMP deaminase but can internalise the plasma protein

Abstract

Histidine-rich glycoprotein (HRG) is synthesized by liver and is present at relatively high concentration in the plasma of vertebrates. We have previously described the association of a HRG-like molecule to purified rabbit skeletal muscle AMP deaminase (AMPD). We also provided the first evidence for the presence of a HRG-like protein in human skeletal muscle where a positive correlation between HRG content and total determined AMPD activity has been shown. In the present paper we investigate the origin of skeletal muscle HRG. The screening of a human skeletal muscle cDNA expression library using an anti-HRG antibody failed to reveal any positive clone. The RT-PCR analysis, performed on human skeletal muscle RNA as well as on RNA from the rhabdomyosarcoma (RD) cell line, failed to show any mRNA specific for the plasma HRG or for the putative muscle variant. When the RD cells were incubated with human plasma HRG, a time-dependent increase of the HRG immunoreactivity was detected both at the plasma membrane level and intracellularly. The internalisation of HRG was inhibited by the addition of heparin. The above data strongly suggest that skeletal muscle cells do not synthesize the muscle variant of HRG but instead can actively internalise it from plasma.

Figure 4

Figure 1

RT-PCR analysis. The indicated sizes of the amplified products correspond to the full length and to the 974–1377 and 1540–1807 regions of HRG cDNA. A control PCR for β-actin is also shown. L, human liver (control); SM, human skeletal muscle; RD, RD cell line.

Figure 2

Internalisation of plasma HRG by RD cells. RD cells were incubated in a medium containing human plasma HRG, fixed in 3.6% paraformaldehyde and submitted to immunocytochemistry performed on permeabilised or unpermeabilised cells, as described under Materials and Methods. The pictures show permeabilized RD cells incubated in the presence of 200 µg/mL HRG for 15 min (a), 30 min (b) and 4 h (c), or in the presence of 400 µg/mL HRG for 30 min (e) and 4 h (f). The inserts of panels (a) and (c) show the immunoreactivity of unpermeabilized RD cells incubated in the presence of 200 µg/mL HRG for 15 min and 4 h, respectively. (d) permeabilized RD cells incubated for 4 h in the absence of HRG. Scale bars: 25 µm.

Figure 3

Effect of heparin on the internalisation of HRG by RD cells. Cells were incubated with 200 µg/mL plasma HRG, in the absence (▪) or in the presence (□) of heparin (200 µg/mL), as described under Materials and Methods. The histograms represent the average number of immunoreactive cells, calculated from the observation of 500 randomly selected cells from each sample. Error bars represents the standard error. Differences were considered significant at P<0.01. *P=0.004.