Biol. Chem. 2021; 402(9): 1073–1085 Martha Paluschinski, Cheng Jun Jin, Natalia Qvartskhava, Boris Görg, Marianne Wammers, Judith Lang, Karl Lang, Gereon Poschmann, Kai Stühler and Dieter Häussinger* Characterization of the scavenger cell proteome in mouse and rat liver + https://doi.org/10.1515/hsz-2021-0123 The data suggest that the population of perivenous GS Received January 25, 2021; accepted July 4, 2021; scavenger cells is heterogeneous and not uniform as previ- published online July 30, 2021 ously suggested which may reflect a functional heterogeneity, possibly relevant for liver regeneration. Abstract: The structural-functional organization of ammonia and glutamine metabolism in the liver acinus involves highly Keywords: glutaminase; glutamine synthetase; liver specialized hepatocyte subpopulations like glutamine syn- zonation; proteomics; scavenger cells. thetase (GS) expressing perivenous hepatocytes (scavenger cells). However, this cell population has not yet been char- acterized extensively regarding expression of other genes and Introduction potential subpopulations. This was investigated in the present study by proteome profiling of periportal GS-negative and There is a sophisticated structural-functional organization in perivenous GS-expressing hepatocytes from mouse and rat. the liver acinus with regard to ammonium and glutamine Apart from established markers of GS+ hepatocytes such as metabolism (Frieg et al. 2021; Gebhardt and Mecke 1983; glutamate/aspartate transporter II (GLT1) or ammonium Häussinger 1983, 1990). Periportal hepatocytes express en- transporter Rh type B (RhBG), we identified novel scavenger zymes required for urea synthesis such as the rate-controlling cell-specific proteins like basal transcription factor 3 (BTF3) enzyme carbamoylphosphate synthetase 1 (CPS1) and liver- and heat-shock protein 25 (HSP25). Interestingly, BTF3 and type glutaminase 2 (GLS2) (for review see Häussinger (1990)). HSP25 were heterogeneously distributed among GS+ hepato- GLS2 is activated by its product ammonium and therefore fi cytes in mouse liver slices. Feeding experiments showed that acts as a pH-regulated mitochondrial ammonium ampli er RhBG expression was increased in livers from mice fed with (Häussinger 1983; Häussinger and Sies 1979; Häussinger fi fi high protein diet compared to standard chow. While spatial et al. 1984). This ampli cation is required for ef cient distributions of GS and carbamoylphosphate synthetase 1 ammonium elimination via urea synthesis in view of the low fi (CPS1) were unaffected, periportal areas constituted by af nity of CPS1 for ammonia and the physiologically low glutaminase 2 (GLS2)-positive hepatocytes were enlarged or ammonium ion concentrations in the portal blood (for review reduced in response to high or low protein diet, respectively. see Häussinger (1990)). Whereas periportal urea synthesis reflects a high capacity, but low affinity-system for ammo- nium disposal, ammonia escaping periportal urea synthesis *Corresponding author: Dieter Häussinger, Clinic for reaches a small perivenous cell population at the acinar Gastroenterology, Hepatology and Infectiology, Heinrich Heine outflow, which removes ammonium ions with high affinity University, Universitätsstr. 1, 40225 Düsseldorf, Germany, through glutamine synthesis. These GS+ hepatocytes were E-mail: [email protected] Martha Paluschinski, Cheng Jun Jin, Natalia Qvartskhava, Boris Görg also called perivenous ‘scavenger cells’, because they and Marianne Wammers, Clinic for Gastroenterology, Hepatology and remove not only ammonium ions, but also other compounds Infectiology, Heinrich Heine University, Universitätsstr. 1, 40225 with high affinity, before the sinusoidal blood reaches the Düsseldorf, Germany. https://orcid.org/0000-0002-6899-3753 hepatic veins (Häussinger 1990; Häussinger and Stehle (M. Paluschinski). https://orcid.org/0000-0002-4630-9420 (B. Görg) 1988). These scavenger cells exclusively express not only Judith Lang and Karl Lang, Institute of Immunology, Medical Faculty, University of Duisburg-Essen, Hufelandstr. 55, 45122 Essen, Germany glutamine synthetase (GS) in the liver but also proteins Gereon Poschmann, Institute of Molecular Medicine, Proteome supporting glutamine synthesis, such as the glutamate/ Research, Medical Faculty, Heinrich Heine University, Universitätsstr. aspartate transporter II (GLT1), ornithine aminotransferase 1, 40225 Düsseldorf, Germany (OAT1), the ammonium transporter Rh type B (RhBG) or Kai Stühler, Institute of Molecular Medicine, Proteome Research, uptake systems for dicarboxylates (Boon et al. 1999; Häus- Medical Faculty, Heinrich Heine University, Universitätsstr. 1, 40225 singer and Gerok 1983; Stoll and Häussinger 1991; Weiner Düsseldorf, Germany; and Molecular Proteomics Laboratory (MPL), Biomedical Research Center (BMFZ), Heinrich Heine University, et al. 2003). The important role of these perivenous + Universitätsstr. 1, 40225 Düsseldorf, Germany GS (scavenger) hepatocytes for ammonium homeostasis is Open Access. © 2021 Martha Paluschinski et al., published by De Gruyter. This work is licensed under the Creative Commons Attribution 4.0 International License. 1074 M. Paluschinski et al.: Proteomic profiling of hepatic scavenger cells underlined by the finding that deletion of GS in mouse liver express the liver-type glutaminase (GLS2). For this, scav- triggers systemic hyperammonemia (Qvartskhava et al. enger cells were labeled using antibodies directed against 2015). Hyperammonemia was also observed in taurine the glutamate transporter 1 (GLT1) (Cadoret et al. 2002), transporter knockout mice, which exhibit impaired peri- while periportal GLS2-expressing hepatocytes were labeled venous glutamine synthesis due to an inactivating tyrosine using antibodies directed against the periportal hepatocyte nitration of GS and downregulation of RhBG (Qvartskhava marker E-cadherin (E-Cad) (Hempel et al. 2015). Moreover, et al. 2019). we investigated effects of dietary protein load on the levels Earlier studies suggest that metabolic liver zonation is of GS and GLS2 in the liver. not static, but rather dynamic and may change in response Our study identifies new proteins being enriched in to nutrients, metabolites or hormones and under patho- perivenous scavenger cells and gives evidence for cell logical conditions such as liver cirrhosis or hepatocellular heterogeneities among GS+ scavenger cells. carcinoma (Boon et al. 1999; Gebhardt and Matz-Soja 2014; Jungermann 1995). Two recent studies reported spatial transcriptome Results profiles in mouse liver (Ben-Moshe et al. 2019; Halpern et al. 2017) and established a detailed gene landscape Distribution of ammonium metabolism- across the liver acinus in spatially defined areas by means related proteins in rodent livers of single-cell sequencing. Due to limited resolution, cellular heterogeneity within a defined area was not taken The distribution of GS, GLS2, CPS1, GLT1 and RhBG protein into account in these studies (Ben-Moshe et al. 2019; Hal- in liver sections was investigated by immunofluorescence pern et al. 2017). analyses (Figure 1). GLS2+ hepatocytes were confined to the In the present study, we characterized the proteome of periportal zone, GS+ scavenger cells surrounded the central perivenous GS+ scavenger cells and compared it to the vein and both subpopulations were clearly demarked by a proteome of GS− hepatocytes (which include periportal and mid-zone constituted by GLS2−/GS− hepatocytes in rodent midzonal hepatocytes) from both, mouse and rat liver. In a liver slices. CPS1 was found in GLS2+ and in hepatocytes of second approach, we compared the proteome of peri- the transitional zone but not in GS+ scavenger cells. As venous scavenger cells and periportal hepatocytes who shown in Figure 1, GS strongly colocalized with RhBG as Figure 1: Metabolic zonation of the liver. Immunofluorescence analyses of glutamine synthetase (GS), glutaminase 2 (GLS2), carbamoylphosphate synthetase 1 (CPS1), glutamate/ aspartate transporter II (GLT1) and ammonium transporter Rh type B (RhBG) protein in (A) mouse and (B) rat liver sections. Cell nuclei were counterstained with Hoechst 34580. M. Paluschinski et al.: Proteomic profiling of hepatic scavenger cells 1075 well as with GLT1 in scavenger cells from both, rat and As expected, scavenger cell markers such as GS, GLT1 mice. These data suggest that the localization of the and ornithine aminotransferase (OAT), were detected in ammonium metabolism-related proteins GLS2, CPS1 and higher abundances in GS+ hepatocytes (Figure 2C, left GS is similar in mouse and rat livers. Furthermore, the clear panel). Moreover, proteins characteristic for periportal and specific labeling of perivenous scavenger cells by the hepatocytes such as mitochondrial ornithine carbamoyl- antibodies directed against GLT1 and GS confirmed their transferase (OTC), argininosuccinate synthase (ASS1) and suitability for isolating perivenous scavenger cells from the phosphoenolpyruvate carboxykinase (PCK1) were only liver. barely detected in this cell population (Figure 2C, left panel). Interestingly, heat shock protein 25 (HSP25), basal transcription factor 3 (BTF3) and RNAse 4 were higher Characterization of the cellular proteome of abundant in GS+ HCs compared to GS− mouse hepato- scavenger cells from rat and mouse livers cytes. Further proteins showing higher abundances in GS+ scavenger hepatocytes, are shown in Figure 2B and For the characterization of the proteome of GS-expressing Table 1. scavenger cells in rat and mouse