Document Detail


Cloning and expression of human sialic acid pathway genes to generate CMP-sialic acids in insect cells.
MedLine Citation:
PMID:  11602804     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
The addition of sialic acid residues to glycoproteins can affect important protein properties including biological activity and in vivo circulatory half-life. For sialylation to occur, the donor sugar nucleotide cytidine monophospho-sialic acid (CMP-SA) must be generated and enzymatically transferred to an acceptor oligosaccharide. However, examination of insect cells grown in serum-free medium revealed negligible native levels of the most common sialic acid nucleotide, CMP-N-acetylneuraminic acid (CMP-Neu5Ac). To increase substrate levels, the enzymes of the metabolic pathway for CMP-SA synthesis have been engineered into insect cells using the baculovirus expression system. In this study, a human CMP-sialic acid synthase cDNA was identified and found to encode a protein with 94% identity to the murine homologue. The human CMP-sialic acid synthase (Cmp-Sas) is ubiquitously expressed in human cells from multiple tissues. When expressed in insect cells using the baculovirus vector, the encoded protein is functional and localizes to the nucleus as in mammalian cells. In addition, co-expression of Cmp-Sas with the recently cloned sialic acid phosphate synthase with N-acetylmannosamine feeding yields intracellular CMP-Neu5Ac levels 30 times higher than those observed in unsupplemented CHO cells. The absence of any one of these three components abolishes CMP-Neu5Ac production in vivo. However, when N-acetylmannosamine feeding is omitted, the sugar nucleotide form of deaminated Neu5Ac, CMP-2-keto-3-deoxy-D-glycero-D-galacto-nononic acid (CMP-KDN), is produced instead, indicating that alternative sialic acid glycoforms may eventually be possible in insect cells. The human CMP-SAS enzyme is also capable of CMP-N-glycolylneuraminic acid (CMP-Neu5Gc) synthesis when provided with the proper substrate. Engineering the CMP-SA metabolic pathway may be beneficial in various cell lines in which CMP-Neu5Ac production limits sialylation of glycoproteins or other glycans.
Authors:
S M Lawrence; K A Huddleston; N Tomiya; N Nguyen; Y C Lee; W F Vann; T A Coleman; M J Betenbaugh
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Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't; Research Support, U.S. Gov't, Non-P.H.S.    
Journal Detail:
Title:  Glycoconjugate journal     Volume:  18     ISSN:  0282-0080     ISO Abbreviation:  Glycoconj. J.     Publication Date:  2001 Mar 
Date Detail:
Created Date:  2001-10-16     Completed Date:  2002-03-04     Revised Date:  2006-11-15    
Medline Journal Info:
Nlm Unique ID:  8603310     Medline TA:  Glycoconj J     Country:  United States    
Other Details:
Languages:  eng     Pagination:  205-13     Citation Subset:  IM    
Affiliation:
Department of Chemical Engineering, The Johns Hopkins University, Baltimore, MD 21218, USA.
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MeSH Terms
Descriptor/Qualifier:
Amino Acid Sequence
Animals
Baculoviridae / genetics,  metabolism
CHO Cells
Cell Fractionation
Cell Line
Cloning, Molecular*
Cricetinae
Cytidine Monophosphate / metabolism*
Cytidine Monophosphate N-Acetylneuraminic Acid / metabolism*
Humans
Molecular Sequence Data
N-Acylneuraminate Cytidylyltransferase / chemistry,  genetics*,  metabolism*
Sequence Alignment
Sialic Acids / genetics,  metabolism*
Spodoptera / genetics
Chemical
Reg. No./Substance:
0/Sialic Acids; 3063-71-6/Cytidine Monophosphate N-Acetylneuraminic Acid; 63-37-6/Cytidine Monophosphate; EC 2.7.7.43/N-Acylneuraminate Cytidylyltransferase

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine


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