Occurrence of a multimeric high-molecular-weight glyceraldehyde-3-phosphate dehydrogenase in human serum.
Cellular glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a phylogenetically conserved, ubiquitous enzyme that plays an indispensable role in energy metabolism. Although a wealth of information is available on cellular GAPDH, there is a clear paucity of data on its extracellular counterpart (i.e., the secreted or extracellular GAPDH). Here, we show that the extracellular GAPDH in human serum is a multimeric, high-molecular-weight, yet glycolytically active enzyme. The high-molecular-weight multimers of serum GAPDH were identified by immunodetection on one- and two-dimensional gel electrophoresis using multiple antibodies specific for various epitopes of GAPDH. Partial purification of serum GAPDH by DEAE Affigel affinity/ion exchange chromatography further established the multimeric composition of serum GAPDH. In vitro data demonstrated that human cell lines secrete a multimeric, high-molecular-weight enzyme similar to that of serum GAPDH. Furthermore, LC-MS/MS analysis of extracellular GAPDH from human cell lines confirmed the presence of unique peptides of GAPDH in the high-molecular-weight subunits. Furthermore, data from pulse-chase experiments established the presence of high-molecular-weight subunits in the secreted, extracellular GAPDH. Taken together, our findings demonstrate the presence of a high-molecular-weight, enzymatically active secretory GAPDH in human serum that may have a hitherto unknown function in humans.
†Russell H. Morgan Department of Radiology and Radiological Sciences, Johns Hopkins University School of Medicine, Baltimore, Maryland 21287, United States.
Rani
R
Kunjithapatham
†Russell H. Morgan Department of Radiology and Radiological Sciences, Johns Hopkins University School of Medicine, Baltimore, Maryland 21287, United States.
Jean-Francois
JF
Geschwind
‡Mass Spectrometry and Proteomics Facility, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, United States.
Lauren
L
Devine
‡Mass Spectrometry and Proteomics Facility, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, United States.
Tatiana N
TN
Boronina
‡Mass Spectrometry and Proteomics Facility, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, United States.
Robert N
RN
O'Meally
‡Mass Spectrometry and Proteomics Facility, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, United States.
Robert N
RN
Cole
§Department of Pathology, Johns Hopkins University School of Medicine, Baltimore, Maryland 21231, United States.
Michael S
MS
Torbenson
†Russell H. Morgan Department of Radiology and Radiological Sciences, Johns Hopkins University School of Medicine, Baltimore, Maryland 21287, United States.
Shanmugasundaram
S
Ganapathy-Kanniappan
D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D002852:Chromatography, Ion Exchange; D002853:Chromatography, Liquid; D015180:Electrophoresis, Gel, Two-Dimensional; D045604:Extracellular Fluid / Q000201:enzymology*; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000032:analysis* / Q000235:genetics; D006801:Humans; D008322:Mammals; D008969:Molecular Sequence Data; D008970:Molecular Weight; D055503:Protein Multimerization; D044967:Serum / Q000201:enzymology*; D053719:Tandem Mass Spectrometry
D005987:Glyceraldehyde-3-Phosphate Dehydrogenases
2D gel electrophoresis; DEAE Affigel Blue column chromatography; extracellular GAPDH; multimer; serum GAPDH