Department of Life Sciences, Ben Gurion University, PO Box 653, Beersheva 84105, Israel
Correspondence
jeichler{at}bgumail.bgu.ac.il
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ABSTRACT |
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Overview |
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Molecular adaptations of archaeal S-layer (glyco)proteins to life in extreme environments |
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Haloarchaeal S-layer glycoproteins
While S-layer (glyco)proteins have been identified in archaea living in a wide range of physical conditions, the best characterized are the S-layer glycoproteins of halophilic archaea. To date, three haloarchaeal S-layer glycoproteins have been studied in detail, i.e. the S-layer glycoproteins of Halobacterium salinarum (Lechner & Sumper, 1987), Haloferax volcanii (Sumper et al., 1990
) and Haloarcula japonica (Wakai et al., 1997
). As with many haloarchaeal proteins, haloarchaeal S-layer glycoproteins are enriched in acidic residues relative to their non-halophilic counterparts. An enhanced number of acidic residues is thought to encourage proper protein folding in high-salt conditions (Madern et al., 2000
; Fukuchi et al., 2003
). In each case, the S-layer glycoproteins are believed to be anchored to the plasma membrane via a single-transmembrane domain located near the C-terminus. The three haloarchaeal S-layer glycoproteins share significant sequence similarity throughout their lengths, except as one approaches the N-terminus. Such sequence divergence does not, however, seem to translate into structural differences, given the inability of computer-aided reconstructions to detect any significant dissimilarities between the Halobacterium salinarum and Haloferax volcanii S-layers (Trachtenberg et al., 2000
). In terms of glycosylation, haloarchaeal S-layer glycoproteins undergo both N- and O-glycosylation (Lechner & Sumper, 1987
; Sumper et al., 1990
). In the Halobacterium salinarum and Haloferax volcanii S-layer glycoproteins, N-glycosylation sites are distributed throughout the length of the sequence, whereas O-glycosylation sites are clustered in a threonine-rich region immediately preceding the C-terminal membrane-spanning domain. Despite overall similarities at the amino acid level, striking differences in N-glycosylation are, however, observed across strain lines, and are thought to reflect differences in the salinity encountered by each strain (Mengele & Sumper, 1992
). Whereas only neutral oligosaccharides are present in the S-layer glycoprotein of Haloferax volcanii, considered a moderate halophile (optimal growth at 1·72·5 M NaCl), the S-layer glycoprotein in the extreme halophile Halobacterium salinarum (optimal growth at 34 M NaCl) contains sulfated glucuronic acid residues and a higher degree of glycosylation. It is thought that these differences, leading to an increased density in surface charges in the latter strain, reflect an adaptation in response to the higher salt concentrations experienced by Halobacterium salinarum. In the Haloarcula japonica S-layer glycoprotein, five N-glycosylation sites, clustered towards the C-terminus, are predicted, although the character of the putative oligosaccharide has yet to be described (Wakai et al., 1997
).
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Biogenesis of archaeal S-layer (glyco)proteins |
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Glycosylation
The S-layer glycoprotein of Halobacterium salinarum was the first non-eukaryal glycoprotein to be described in detail (Lechner & Sumper, 1987). Since, the glycan profile of additional archaeal S-layer glycoproteins has been described. Detailed descriptions of the molecular composition of these moieties, their modes of attachment to S-layer proteins as well as other questions related to archaeal saccharide chemistry have been treated in a series of recent reviews (Sumper & Wieland, 1995
; Messner, 1997
; Moens & Vanderleyden, 1997
; Schäffer & Messner, 2001
; Messner & Schäffer, 2003
). Here, general lessons on archaeal protein glycosylation learned through analysis of S-layer protein glycosylation are considered.
In addressing archaeal S-layer glycoprotein biogenesis, species-specific aspects of the archaeal glycosylation process have been revealed. In Sulfolobus acidocaldarius, tunicamycin, known to interfere with the transfer of UDP-N-acetylglucosamine to dolichol phosphate polysaccharide carriers (Elbein, 1981), hinders S-layer glycoprotein glycosylation (Grogan, 1996
). In contrast, tunicamycin has no effect on the biosynthesis of the Haloferax volcanii S-layer glycoprotein (Eichler, 2001
). Similarly, bacitracin, shown to interfere with glycosylation of the Halobacterium salinarum S-layer glycoprotein (Mescher & Strominger, 1976
), also had no effect on Haloferax volcanii S-layer glycoprotein biosynthesis (Eichler, 2001
). In Halobacterium salinarum, bacitracin is thought to interfere with the processing of the dolichyl pyrophosphate carrier used for glycosylation at the Asn-2 position of the S-layer glycoprotein (Wieland et al., 1981
). The failure of the antibiotic to modify Haloferax volcanii S-layer glycoprotein biogenesis is likely related to the fact that, unlike in Halobacterium salinarum, where both monophosphate- and pyrophosphate-linked dolichol oligosaccharide carriers are present (Lechner & Wieland, 1989
), only monophosphate-linked oligosaccharide-dolichol intermediates are detected in Haloferax volcanii (Kuntz et al., 1997
).
Exploring S-layer glycoprotein glycosylation has also revealed the sophistication of the archaeal protein glycosylation machinery. The Halobacterium salinarum S-layer glycoprotein contains two different types of N-linked oligosaccharide chains, with sulfated glucuronic acid moieties attached to asparagine-linked glucose residues predominating and a single chain of a sulfated repeating unit pentasaccharide linked through N-acetylgalactosamine positioned at the 2-asparagine position of the protein (Lechner & Wieland, 1989). It remains unclear how the archaeal glycosylation machinery determines which oligosaccharide entity is to be attached to a particular glycosylation site. Moreover, it could be shown that replacement of the serine residue at position 4 of the protein by a valine, leucine or asparagine residue did not interfere with normal S-layer glycoprotein glycosylation, suggesting that motifs apart from the consensus Asn-Xaa-Ser/Thr sequence are recognized by the haloarchaeal glycosylation machinery (Zeitler et al., 1998
).
Finally, several studies addressing S-layer glycoprotein glycosylation suggest that in archaea, protein glycosylation occurs on the outer cell surface. Despite being unable to permeate the plasma membrane, bacitracin selectively prevented Halobacterium salinarum S-layer glycoprotein glycosylation (Mescher & Strominger, 1976). The ability of Halobacterium salinarum cells to add sulfated oligosaccharides, such as those that decorate the S-layer glycoprotein in this species, to exogenously added cell impermeant hexapeptides containing an N-glycosylation motif further supports the assignment of the haloarchaeal glycosylation machinery to the external cell surface (Lechner & Wieland, 1989
). Similarly, in studies relying on the glucosyltransferase inhibitors amphomycin, PP36 and PP55, it was concluded that glycosylation of Haloferax volcanii glycoproteins, including the S-layer glycoprotein, occurs on the outer cell surface (Zhu et al., 1995
). Hence it appears that glycosylation of haloarchaeal S-layer glycoproteins is topologically homologous to the eukaryal protein glycosylation process, as in both cases translocation across a membrane precedes protein modification. In eukarya, protein glycosylation only begins once a protein has traversed the membrane of the ER (Kornfeld & Kornfeld, 1985
).
Lipid modification
In addition to glycosylation, archaeal S-layer (glyco)proteins may also experience additional post-translational modifications. In studies addressing the biosynthesis of the Haloferax volcanii S-layer glycoprotein, it was reported that the protein is first synthesized as an immature precursor, possessing a lower apparent molecular mass and less hydrophobic character than the final version of the protein (Eichler, 2001). The post-translational conversion to the mature form apparently involves isoprenylation, since the S-layer glycoprotein can be labelled with [3H]mevalonic acid, an isoprene precursor (Konrad & Eichler, 2002
). This observation is in line with earlier findings that the Halobacterium salinarum S-layer glycoprotein is modified by a covalently linked diphytanylglyceryl phosphate moiety (Kikuchi et al., 1999
). Indeed, the Halobacterium salinarum S-layer glycoprotein also undergoes maturation similar to that experienced by its Haloferax volcanii counterpart, suggesting that isoprenylation may be a general feature of haloarchaeal S-layer glycoproteins (Konrad & Eichler, 2002
). A combination of pulsechase radiolabelling and subcellular fractionation approaches has shown that lipid modification of the Haloferax volcanii S-layer glycoprotein only occurs once the S-layer glycoprotein has been delivered across the plasma membrane (Eichler, 2001
). Like protein glycosylation, lipid modification of eukaryal proteins also occurs at the lumenal face of the ER membrane, i.e. once the protein has translocated across the membrane (Wang et al., 1999
). This, therefore, reveals further functional parallels between the archaeal external surface and the lumenal face of the ER membrane. Still, it is not clear why haloarchaeal S-layer glycoproteins would require an isoprene-based link to the membrane in addition to the C-terminal membrane-spanning domain. One possibility would be to offer physical support to the S-layer itself, thereby creating a periplasm-like space (Kessel et al., 1988
; Peters et al., 1995
). Alternatively, lipid modification of membrane-inserted haloarchaeal S-layer glycoproteins could reflect a primitive version of a mode of protein membrane anchoring prevalent in eukarya.
Turnover
In at least one archaeal species, the S-layer includes additional proteins that may be involved in the breakdown of S-layer (glyco)proteins. In Staphylothermus marinus, the S-layer is composed of an S-layer glycoprotein together with a lighter, 85 kDa chain, both originating from the same gene (Peters et al., 1995). In addition, a 130 kDa globular glycoprotein, shown to be a subtilisin-like protease functional at elevated temperatures in the presence of detergent or denaturants, is also present (Mayr et al., 1996
). Although capable of digesting the S-layer glycoprotein, the physiological function of the protease is not clear. It is tempting to speculate that remodelling of the archaeal S-layer via degradation of S-layer proteins is a normal part of various cellular functions involving the S-layer, such as cell division (Pum et al., 1991
), morphological changes (Mayerhofer et al., 1998
) or transfer of genetic material (Rosenshine et al., 1989
; Schleper et al., 1995
).
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Conclusions |
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ACKNOWLEDGEMENTS |
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