Glycobiology. 2015 Dec;25(12):1431-40.

Cloning and expression of 3-deoxy-d-manno-oct-2-ulosonic acid α-ketoside hydrolase from oyster hepatopancreas.

Nakagawa T1, Shimada Y1, Pavlova NV1, Li SC1, Li YT2.
1Department of Biochemistry and Molecular Biology, Tulane University School of Medicine, New Orleans, LA 70112, USA.
2Department of Biochemistry and Molecular Biology, Tulane University School of Medicine, New Orleans, LA 70112, USA yli1@tulane.edu ytlscl@gmail.com.

 

Abstract

We have previously reported that oyster hepatopancreas contained three unusual α-ketoside hydrolases: (i) a 3-deoxy-d-manno-oct-2-ulosonic acid α-ketoside hydrolase (α-Kdo-ase), (ii) a 3-deoxy-D-glycero-D-galacto-non-2-ulosonic acid α-ketoside hydrolase and (iii) a bifunctional ketoside hydrolase capable of cleaving both the α-ketosides of Kdn and Neu5Ac (Kdn-sialidase). After completing the purification of Kdn-sialidase, we proceeded to clone the gene encoding this enzyme. Unexpectedly, we found that instead of expressing Kdn-sialidase, our cloned gene expressed α-Kdo-ase activity. The full-length gene, consisting of 1176-bp (392 amino acids, Mr 44,604), expressed an active recombinant α-Kdo-ase (R-α-Kdo-ase) in yeast and CHO-S cells, but not in various Escherichia coli strains. The deduced amino acid sequence contains two Asp boxes (S(277)PDDGKTW and S(328)TDQGKTW) commonly found in sialidases, but is devoid of the signature FRIP-motif of sialidase. The R-α-Kdo-ase effectively hydrolyzed the Kdo in the core-oligosaccharide of the structurally defined lipopolysaccharide (LPS), Re-LPS (Kdo(2)-Lipid A) from Salmonella minnesota R595 and E. coli D31m4. However, Rd-LPS from S. minnesota R7 that contained an extra outer core phosphorylated heptose was only slowly hydrolyzed. The complex type LPS from Neisseria meningitides A1 and M992 that contained extra 5-6 sugar units at the outer core were refractory to R-α-Kdo-ase. This R-α-Kdo-ase should become useful for studying the structure and function of Kdo-containing glycans.

KEYWORDS:glycosidase; lipid A; recombinant enzyme; sialidase; α-Kdo-ase

PMID:26362869; DOI:10.1093/glycob/cwv074  

Supplement:

Our revelation of the gene encoding oyster a-Kdo-ase can be regarded as serendipitous. We set out to clone the gene encoding the oyster Kdn-sialidase and were frustrated by the unexpected twists and turns of the work. At the end, we were rewarded by the uncovering of the gene that encodes a-Kdo-ase.

 

To understand the structure and function of Kdn and kdo, our laboratory became interested in Kdn-ase (kdn-cleaving sialidase) and a-Kdo-ase in the 1990s. Figure 1 shows the structures of Neu5Ac, Kdn (2-keto-3-deoxy-D-glycero-D-galacto-2-nonulosonic acid) and Kdo (2-keto-3-deoxy-D-manno-oct-2-ulosonic acid). These three monosaccharides are all 2-keto-3-deoxy sugars with a carboxylic acid at the C1 position. Neu5Ac and Kdn are both nine-carbon acidic sugars, while Kdo is an eight-carbon acidic sugar. The only difference between Neu5Ac and Kdn is that at C5 position Neu5Ac has an -NHAc, while Kdn has an -OH. As shown in Figure 1, Kdo can be regarded as a one-carbon truncated form of Kdn.  However, in Kdo, the configurations of the -OH residues at C4 and C6 are different from that of Neu5Ac and Kdn. From the conformational point of view, Kdo is distinct from Neu5Ac and Kdn. The cyclic form of Kdo is in the 5C2 conformation, whereas both Kdn and Neu5Ac are in the 2C5 conformation. Nevertheless, all three sugars use CMP-sugar donors (CMP-Kdn, CMP-Neu5Ac, and CMP-Kdo) for their biosynthesis of glycans. Thus, Kdo has been regarded as an extended member of the sialic acid family (1).

Although Kdn and Kdo are structurally related, their occurrence and function are quite different. Kdn has been found in the glycans of mammalian tissues and marine organisms (2). Kdo is a highly conserved sugar component found in the inner core of lipid A of the Gram-negative bacterial lipopolysaccharide (LPS) and lipid A with Kdo domain is required for the growth of Gram-negative bacteria. Further modifications of lipid A with additional Kdo, heptoses, and oligosaccharides are associated with the expression of various bacterial serotypes, the antibody types against the specific chemical structures (chemotypes) of the bacterial LPS (3). Kdo also exists in the plant cell wall rhamnogalacturonans that are different from the lipid A-like molecules (4).

 

To work on a-Kdn-ase and a-Kdo-ase in the 1990s, our first step was to synthesize the fluorogenic substrates, 4-methylumbelliferyl (MU)-a-Kdn (5) and MU-a-Kdo (6), since they were not commercially available at that time.

 

Oysters are filter feeders living in estuaries. They consume plankton, bacteria, algae and other organic particles as their food sources. In addition to being found in bacteria, Kdo was also found in algae (7). Based on their food intake, we reasoned that oyster should contain a-Kdo-ase to digest Kdo-containing glycoconjugates in marine organisms and we carried out the purification and characterization of α-Kdo-ase from the hepatopancreas of oyster, Crassostrea virginica, in 1997 (6). In 1999, we subsequently found that oyster hepatopancreas was devoid of the conventional sialidase, but contained two Kdn-cleaving sialidases (8). The major one, Kdn-sialidase, effectively cleaved a-linked Kdn and also slowly hydrolyzed a-linked Neu5Ac. The minor one, Kdn-ase, exclusively cleaved a-linked Kdn. For the purpose of cloning Kdn-sialidase, we purified oyster Kdn-sialidase to electrophoretically homogeneous form (8) and obtained three tryptic peptides: Peptide 1, SGDSAEIWVLSAR; Peptide 2, NTWLYPIYYAGGSSQEQTSNLK; and Peptide 3, GQPHLSAFFR. In addition, we found that the N-terminus of the native enzyme protein was identical to Peptide 1.

 

Using the degenerate oligonucleotide probes deduced from these three peptides, we followed the conventional cloning procedures to obtain a cDNA clone expected to encode Kdn-sialidase. We proceeded to express this gene in E. coli first and then in yeast and CHO-S cells. However, we were not able to detect any Kdn-sialidase activity under a variety of conditions. After struggling for over two years, we reconsidered the structural similarity of Kdo and Kdn. Since oyster hepatopancreas also contained Kdo-ase, we decided to check the expressed protein for Kdo-ase activity. We were pleasantly surprised that this expressed protein, presumed to be Kdn-sialidase, had a very strong a-Kdo-cleaving activity in yeast and CHO-S cells but not in E. coli. Thus, our expressed protein was an a-Kdo-ase. This surprising result could be due to one of the two possibilities. One is that Kdn-sialidase and a-Kdo-ase might have very similar chemical and physical properties and the two enzymes might migrate at the close proximity on SDS gel electrophoresis. Because of this, the tryptic peptides that generated from the excised gel-band might be derived from the native α-Kdo-ase. The other is that the gene encoding Kdn-sialidase might have high homology to that encoding α-Kdo-ase. The PCR-cloning strategies using the degenerate primers designed based on the three tryptic-peptides of the native Kdn-sialidase might have enriched the homologous DNA sequence that encodes α-Kdo-ase. Only the revelation of the gene that encodes oyster Kdn-sialidase can we verify which of the above two possibilities resulted in our unexpected finding.

 

The availability of α-Kdo-ase would facilitate the study of the structures and functions of α-Kdo containing glycans.

 

References

  1. Chen X, Varki A (2010). Advances in the biology and chemistry of sialic acids. ACS Chem Biol. 5:163-176.
  2. Inoue S, Kitajima K (2006). KDN (deaminated neuraminic acid): dreadful past and exciting future of the newest member of the sialic acid family. Glycoconj J 23:277-290.
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  5. Li YT, Yuziuk JA, Li SC, Nematalla A, Hasegawa A, Kimura M, and Nakagawa H (1994). A novel sialidase capable of cleaving 3-deoxy-D-glycero-D-galacto-2-nonulosonic acid (KDN). Arch Biochem Biophys. 310: 243-246.
  6. Li YT, Wang LX, Pavlova NV, Li SC, Lee YC (1997). a-KDOase activity in oyster and synthesis of a- and b-4-methylumbelliferyl ketosides of 3-deoxy-D-mannooctulosonic acid (KDO). J Biol Chem. 272: 26419-26424.
  7. Becker B, Lommerse JPM, Melkonian M, Kamerling JP, Vliegenthart JFG (1995). The structure of an acidic trisaccharide component from a cell wall polysaccharide preparation of the green alga Tetraselmis striata Butcher. Carbohydr Res 267:313-321.
  8. Pavlova NV, Yuziuk JA, Nakagawa H, Kiso M, Li SC, and Li, YT (1999) 2-Keto-3-deoxy-D-glycero-D-galacto-nononic acid (KDN)- and N-acetylneuraminic acid-cleaving sialidase (KDN-sialidase) and KDN-cleaving hydrolase (KDNase) from the hepatopancreas of oyster, Crassostrea virginica. J Biol Chem. 274:31974-31980.

 

 

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