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Hyaluronan Synthase: The Mechanism of Initiation at the Reducing End and a Pendulum Model for Polysaccharide Translocation to the Cell Exterior.

Weigel PH - Int J Cell Biol (2015)

Bottom Line: Class I family members include mammalian and streptococcal HASs, the focus of this review, which add new intracellular sugar-UDPs at the reducing end of growing hyaluronyl-UDP chains.The synthesis of chitin-UDP oligomers by HAS confirms the reducing end mechanism for sugar addition during HA assembly by streptococcal and mammalian Class I enzymes.These new findings indicate the possibility that HA biosynthesis is initiated by the ability of HAS to use chitin-UDP oligomers as self-primers.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry & Molecular Biology, The Oklahoma Center for Medical Glycobiology, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73190, USA.

ABSTRACT
Hyaluronan (HA) biosynthesis has been studied for over six decades, but our understanding of the biochemical details of how HA synthase (HAS) assembles HA is still incomplete. Class I family members include mammalian and streptococcal HASs, the focus of this review, which add new intracellular sugar-UDPs at the reducing end of growing hyaluronyl-UDP chains. HA-producing cells typically create extracellular HA coats (capsules) and also secrete HA into the surrounding space. Since HAS contains multiple transmembrane domains and is lipid-dependent, we proposed in 1999 that it creates an intraprotein HAS-lipid pore through which a growing HA-UDP chain is translocated continuously across the cell membrane to the exterior. We review here the evidence for a synthase pore-mediated polysaccharide translocation process and describe a possible mechanism (the Pendulum Model) and potential energy sources to drive this ATP-independent process. HA synthases also synthesize chitin oligosaccharides, which are created by cleavage of novel oligo-chitosyl-UDP products. The synthesis of chitin-UDP oligomers by HAS confirms the reducing end mechanism for sugar addition during HA assembly by streptococcal and mammalian Class I enzymes. These new findings indicate the possibility that HA biosynthesis is initiated by the ability of HAS to use chitin-UDP oligomers as self-primers.

No MeSH data available.


Membrane organization of HAS domains and conserved potential glycosyl-UDP binding regions. The experimentally determined topology of SpHAS [28] is modified to incorporate the discovery [46] that all four Cys residues of SeHAS (white circles) are at the membrane-protein interface and are located in or very near to the sugar-UDP binding sites. These four Cys residues are positionally conserved in the Class I HAS family. The SeHAS numbering shows the amino acids at the cytoplasmic junctions of the six MDs (white numbers 1–6). The parallel lines (gray) between C262 and C281 indicate the close proximity (~5 A) of these residues; they are not disulfide bonded. Eight “DXD”- or “XDD”-equivalent motifs in SeHAS, potential glycosyl-UDP binding sites (rectangle boxes), are either conserved just among the streptococcal enzymes (light gray) or also among the eukaryotic HASs (white); a few exceptions are discussed in the text. In some motifs, a streptococcal acidic residue is shaded white to indicate its conservation in the HAS family.
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fig2: Membrane organization of HAS domains and conserved potential glycosyl-UDP binding regions. The experimentally determined topology of SpHAS [28] is modified to incorporate the discovery [46] that all four Cys residues of SeHAS (white circles) are at the membrane-protein interface and are located in or very near to the sugar-UDP binding sites. These four Cys residues are positionally conserved in the Class I HAS family. The SeHAS numbering shows the amino acids at the cytoplasmic junctions of the six MDs (white numbers 1–6). The parallel lines (gray) between C262 and C281 indicate the close proximity (~5 A) of these residues; they are not disulfide bonded. Eight “DXD”- or “XDD”-equivalent motifs in SeHAS, potential glycosyl-UDP binding sites (rectangle boxes), are either conserved just among the streptococcal enzymes (light gray) or also among the eukaryotic HASs (white); a few exceptions are discussed in the text. In some motifs, a streptococcal acidic residue is shaded white to indicate its conservation in the HAS family.

Mentions: SpHAS, the only Class I HAS whose topology has been determined experimentally [28], has the N- and C-terminus and majority of the SpHAS protein inside the cell (Figure 2). StrepHASs have six membrane domains (MDs), four of which pass through the membrane giving two small loops of the protein exposed to the extracellular side. The other two MDs, one within the large central catalytic domain and one in the C-terminal one-third of the protein, interact with the membrane as amphipathic helices or reentrant loops but do not appear to span the membrane. The presence of two MDs in HAS that are amphipathic and do not cross the membrane is intriguing because these might be particularly well suited for the formation of an intraprotein pore. Vertebrate HASs contain an additional C-terminal region of ~130–160 aa with two trans-MDs. The amino ~75% of the larger eukaryotic HAS family members is homologous to SpHAS with the same predicted domain organization, so the overall topological organization of all the Class I HASs is predicted to be similar [10].


Hyaluronan Synthase: The Mechanism of Initiation at the Reducing End and a Pendulum Model for Polysaccharide Translocation to the Cell Exterior.

Weigel PH - Int J Cell Biol (2015)

Membrane organization of HAS domains and conserved potential glycosyl-UDP binding regions. The experimentally determined topology of SpHAS [28] is modified to incorporate the discovery [46] that all four Cys residues of SeHAS (white circles) are at the membrane-protein interface and are located in or very near to the sugar-UDP binding sites. These four Cys residues are positionally conserved in the Class I HAS family. The SeHAS numbering shows the amino acids at the cytoplasmic junctions of the six MDs (white numbers 1–6). The parallel lines (gray) between C262 and C281 indicate the close proximity (~5 A) of these residues; they are not disulfide bonded. Eight “DXD”- or “XDD”-equivalent motifs in SeHAS, potential glycosyl-UDP binding sites (rectangle boxes), are either conserved just among the streptococcal enzymes (light gray) or also among the eukaryotic HASs (white); a few exceptions are discussed in the text. In some motifs, a streptococcal acidic residue is shaded white to indicate its conservation in the HAS family.
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4581545&req=5

fig2: Membrane organization of HAS domains and conserved potential glycosyl-UDP binding regions. The experimentally determined topology of SpHAS [28] is modified to incorporate the discovery [46] that all four Cys residues of SeHAS (white circles) are at the membrane-protein interface and are located in or very near to the sugar-UDP binding sites. These four Cys residues are positionally conserved in the Class I HAS family. The SeHAS numbering shows the amino acids at the cytoplasmic junctions of the six MDs (white numbers 1–6). The parallel lines (gray) between C262 and C281 indicate the close proximity (~5 A) of these residues; they are not disulfide bonded. Eight “DXD”- or “XDD”-equivalent motifs in SeHAS, potential glycosyl-UDP binding sites (rectangle boxes), are either conserved just among the streptococcal enzymes (light gray) or also among the eukaryotic HASs (white); a few exceptions are discussed in the text. In some motifs, a streptococcal acidic residue is shaded white to indicate its conservation in the HAS family.
Mentions: SpHAS, the only Class I HAS whose topology has been determined experimentally [28], has the N- and C-terminus and majority of the SpHAS protein inside the cell (Figure 2). StrepHASs have six membrane domains (MDs), four of which pass through the membrane giving two small loops of the protein exposed to the extracellular side. The other two MDs, one within the large central catalytic domain and one in the C-terminal one-third of the protein, interact with the membrane as amphipathic helices or reentrant loops but do not appear to span the membrane. The presence of two MDs in HAS that are amphipathic and do not cross the membrane is intriguing because these might be particularly well suited for the formation of an intraprotein pore. Vertebrate HASs contain an additional C-terminal region of ~130–160 aa with two trans-MDs. The amino ~75% of the larger eukaryotic HAS family members is homologous to SpHAS with the same predicted domain organization, so the overall topological organization of all the Class I HASs is predicted to be similar [10].

Bottom Line: Class I family members include mammalian and streptococcal HASs, the focus of this review, which add new intracellular sugar-UDPs at the reducing end of growing hyaluronyl-UDP chains.The synthesis of chitin-UDP oligomers by HAS confirms the reducing end mechanism for sugar addition during HA assembly by streptococcal and mammalian Class I enzymes.These new findings indicate the possibility that HA biosynthesis is initiated by the ability of HAS to use chitin-UDP oligomers as self-primers.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry & Molecular Biology, The Oklahoma Center for Medical Glycobiology, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73190, USA.

ABSTRACT
Hyaluronan (HA) biosynthesis has been studied for over six decades, but our understanding of the biochemical details of how HA synthase (HAS) assembles HA is still incomplete. Class I family members include mammalian and streptococcal HASs, the focus of this review, which add new intracellular sugar-UDPs at the reducing end of growing hyaluronyl-UDP chains. HA-producing cells typically create extracellular HA coats (capsules) and also secrete HA into the surrounding space. Since HAS contains multiple transmembrane domains and is lipid-dependent, we proposed in 1999 that it creates an intraprotein HAS-lipid pore through which a growing HA-UDP chain is translocated continuously across the cell membrane to the exterior. We review here the evidence for a synthase pore-mediated polysaccharide translocation process and describe a possible mechanism (the Pendulum Model) and potential energy sources to drive this ATP-independent process. HA synthases also synthesize chitin oligosaccharides, which are created by cleavage of novel oligo-chitosyl-UDP products. The synthesis of chitin-UDP oligomers by HAS confirms the reducing end mechanism for sugar addition during HA assembly by streptococcal and mammalian Class I enzymes. These new findings indicate the possibility that HA biosynthesis is initiated by the ability of HAS to use chitin-UDP oligomers as self-primers.

No MeSH data available.