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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.


Summary of HAS functions required for HA biosynthesis. At least twelve discrete binding and catalytic function are required for Class I HA synthases to create a (GlcNAc)n-UDP self-primer (A, functions 1–5), to initiate HA disaccharide synthesis (B, functions 6 and 7), and to then assemble HA disaccharide units in a continuous manner (C, functions 8–11), while the growing HA-UDP chain is also continuously translocated through the HAS·lipid complex pore to the cell surface or exterior (D, function 12). Two functions for steady-state HA disaccharide synthesis (C) are also used to make (GlcNAc)n-UDP (#2) or the first HA disaccharide (#6).
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figbox1: Summary of HAS functions required for HA biosynthesis. At least twelve discrete binding and catalytic function are required for Class I HA synthases to create a (GlcNAc)n-UDP self-primer (A, functions 1–5), to initiate HA disaccharide synthesis (B, functions 6 and 7), and to then assemble HA disaccharide units in a continuous manner (C, functions 8–11), while the growing HA-UDP chain is also continuously translocated through the HAS·lipid complex pore to the cell surface or exterior (D, function 12). Two functions for steady-state HA disaccharide synthesis (C) are also used to make (GlcNAc)n-UDP (#2) or the first HA disaccharide (#6).

Mentions: We noted above the seven functions that all Class I HAS enzymes possess in order to catalyze the steady-state assembly of GlcNAC(β1,3)GlcUA(β1,4) disaccharides during HA synthesis. The recent discovery that HASs are also able to make oligo-chitosyl-UDP species and that these can serve as self-primers for subsequent HA assembly means that this enzyme family also possesses more binding site and catalysis functions than previously recognized. These functions are summarized in Box 1, using the standard transferase nomenclature, and listed in order of use in the initiation and assembly of HA. The synthesis of chitin-UDP species requires three binding sites with different structural specificities and two transferase activities (Box 1(A), functions 1–5). Initiation of HA synthesis requires making the first disaccharide using a non-HA substrate, so an acceptor site for the GlcUA to be added and a unique transferase activity (used only once for each HA molecule synthesized) are needed (Box 1(B), functions 6 and 7). Subsequent steady-state HA disaccharide synthesis requires seven functions, using two binding sites noted in (A) and (B) (functions 2 and 6), two additional hyaluronyl-UDP species binding sites, and two additional corresponding transferase activities (Box 1(C), functions 8–11). The final function is the translocation activity of HAS, which acts in a continuous manner during HA chain elongation but is listed separately to emphasize its novel and separate nature, as a “spatial” rather than chemical catalytic process (Box 1(D), function 12). Thus, an astounding 12 discrete functions are attributable to Class I HASs in order for these enzymes to initiate, assemble, and transfer the oligo-chitosyl-HA-UDP polysaccharide to the cell exterior; it is not known if released HA chains are still attached to UDP at their reducing ends or if chains are released because this group has been lost and elongation has therefore stopped, resulting in HA release.


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)

Summary of HAS functions required for HA biosynthesis. At least twelve discrete binding and catalytic function are required for Class I HA synthases to create a (GlcNAc)n-UDP self-primer (A, functions 1–5), to initiate HA disaccharide synthesis (B, functions 6 and 7), and to then assemble HA disaccharide units in a continuous manner (C, functions 8–11), while the growing HA-UDP chain is also continuously translocated through the HAS·lipid complex pore to the cell surface or exterior (D, function 12). Two functions for steady-state HA disaccharide synthesis (C) are also used to make (GlcNAc)n-UDP (#2) or the first HA disaccharide (#6).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

figbox1: Summary of HAS functions required for HA biosynthesis. At least twelve discrete binding and catalytic function are required for Class I HA synthases to create a (GlcNAc)n-UDP self-primer (A, functions 1–5), to initiate HA disaccharide synthesis (B, functions 6 and 7), and to then assemble HA disaccharide units in a continuous manner (C, functions 8–11), while the growing HA-UDP chain is also continuously translocated through the HAS·lipid complex pore to the cell surface or exterior (D, function 12). Two functions for steady-state HA disaccharide synthesis (C) are also used to make (GlcNAc)n-UDP (#2) or the first HA disaccharide (#6).
Mentions: We noted above the seven functions that all Class I HAS enzymes possess in order to catalyze the steady-state assembly of GlcNAC(β1,3)GlcUA(β1,4) disaccharides during HA synthesis. The recent discovery that HASs are also able to make oligo-chitosyl-UDP species and that these can serve as self-primers for subsequent HA assembly means that this enzyme family also possesses more binding site and catalysis functions than previously recognized. These functions are summarized in Box 1, using the standard transferase nomenclature, and listed in order of use in the initiation and assembly of HA. The synthesis of chitin-UDP species requires three binding sites with different structural specificities and two transferase activities (Box 1(A), functions 1–5). Initiation of HA synthesis requires making the first disaccharide using a non-HA substrate, so an acceptor site for the GlcUA to be added and a unique transferase activity (used only once for each HA molecule synthesized) are needed (Box 1(B), functions 6 and 7). Subsequent steady-state HA disaccharide synthesis requires seven functions, using two binding sites noted in (A) and (B) (functions 2 and 6), two additional hyaluronyl-UDP species binding sites, and two additional corresponding transferase activities (Box 1(C), functions 8–11). The final function is the translocation activity of HAS, which acts in a continuous manner during HA chain elongation but is listed separately to emphasize its novel and separate nature, as a “spatial” rather than chemical catalytic process (Box 1(D), function 12). Thus, an astounding 12 discrete functions are attributable to Class I HASs in order for these enzymes to initiate, assemble, and transfer the oligo-chitosyl-HA-UDP polysaccharide to the cell exterior; it is not known if released HA chains are still attached to UDP at their reducing ends or if chains are released because this group has been lost and elongation has therefore stopped, resulting in HA release.

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.