Mycobacterial Glycophosphoinositides: the Work of Clinton E. Ballou

Abstract

Biosynthesis of Mannophosphoinositides by Mycobacterium phlei. The Family of Dimannophosphoinositides

(Brennan, P., and Ballou, C. E. (1967) J. Biol. Chem. 242, 3046–3056)

Biosynthesis of Mannophosphoinositides by Mycobacterium phlei. Enzymatic Acylation of the Dimannophosphoinositides

(Brennan, P., and Ballou, C. E. (1968) J. Biol. Chem. 243, 2975–2984)

Clinton Edward Edgerton Ballou was born in King Hill, Idaho in 1923. After graduating from high school, he enrolled in a premed program at Boise Junior College, but his interests quickly turned to chemistry after he dissected a poorly embalmed cat in a comparative anatomy course. During his sophomore year, Ballou transferred to Oregon State College in Corvallis where he became involved in two research projects: the first during his sophomore year synthesizing new antimalarial drugs with Bert Christensen, and the second during his senior year studying the guinea pig “antistiffness factor” with Willem van Wagtendonk.

Clinton E. Ballou

The military draft was in effect as Ballou entered his last year of college so he decided to join the U. S. Navy after graduating in 1944. He was discharged 2 years later and decided to apply for graduate study in biochemistry with Karl Paul Link at the University of Wisconsin-Madison. As detailed in a previous Journal of Biological Chemistry (JBC) Classic (1), Link's research centered on blood anticoagulants, and when Ballou arrived in his laboratory in 1946, the primary focus was the structure-function relationship of coumarin anticoagulants. Ballou was immediately intrigued when he learned of a failed attempt to synthesize the glucoside of dicumarol because the acetylated intermediate was degraded in the alkali conditions used for deacetylation. Because glycosides are acetals, which are typically acid-labile and alkali-stable, Ballou decided to study a variety of synthetic compounds to try to understand the structural basis for alkali sensitivity. This research formed the core of his doctoral dissertation, and his exposure to carbohydrate chemistry influenced the direction of his career.

After earning his Ph.D. in 1950, Ballou did a year-long postdoctoral fellowship with E. L. Hirst in the Department of Chemistry at the University of Edinburgh. There he studied the structure of maple sapwood starch. At the end of the year, Ballou returned to the U. S. to work with Hermann O. L. Fischer (the son of Nobel laureate Hermann Emil Fischer) at the University of California, Berkeley. Ballou explained his choice: “I was attracted to Fischer in part because of his research on phosphorylated sugars but also because during graduate school I had drawn heavily on the published works of his father, Emil Fischer. I guess the idea of being associated with the son of Emil Fischer just seemed ‘real cool’ to me” (2).

The 1950s was a time of active research on biosynthetic pathways involving short chain phosphorylated sugars, and collaborating with Fischer and Donald MacDonald, Ballou undertook the syntheses of several such metabolic intermediates, including d-glyceric acid 2-phosphate, d-glyceraldehyde 3-phosphate, dihydroxyacetone phosphate, hydroxypyruvic acid 3-phosphate, and d-erythrose 4-phosphate. He also became interested in inositol chemistry as a result of studies on the cyclitols in sugar pine heartwood.

In 1955, Ballou was appointed to the biochemistry faculty at Berkeley and went about setting up an independent research program. He decided to work on inositol-containing phospholipids and was able to synthesize and characterize d-myoinositol 1-phosphate. He also spent several years isolating and characterizing myoinositol polyphosphates from beef brain phosphoinositide. This culminated in his discovery of d-myoinositol l,4,5-trisphosphate or IP₃. More information on Ballou's studies of these phosphoinositides can be found in his JBC Reflections (2).

Ballou became eligible for sabbatical leave in 1961 and decided to spend a year at the National Center for Scientific Research (CNRS) in France studying the glycophosphoinositides of mycobacteria with Edgar Lederer. There he collaborated with Erna Vilkas on experiments to establish the linkages of both the phosphatidyl and the mannosyl groups to the myoinositol ring (3). Upon his return to Berkeley, Ballou and Yuan Chuan Lee determined the structures of the family of mannosyl phosphoinositides in Mycobacterium smegmatis (4–6). Ballou and his postdoctoral fellow Patrick Brennan then began to look at the biosynthesis of the intact dimannophosphoinositides on Mycobacterium phlei, which is the subject of the two JBC Classics reprinted here.

In the first Classic, Ballou and Brennan used subcellular fractions of M. phlei to catalyze the biosynthesis of several mannosyl derivatives of phosphatidylmyoinositol and reported that the major products are a family of three dimannophosphoinositides (A, B, and C) that differ in the number of fatty acyl groups they contain (four, three, and two, respectively). On the basis of their results, they proposed that guanosine diphosphate mannose acts as the sugar donor in the conversion of phosphatidylmyoinositol to phosphatidylmyoinositol dimannoside (dimannophosphoinositide C), which is then acylated in a two-step process to first yield dimannophosphoinositide B and then dimannophosphoinositide A.

In the second JBC Classic, Ballou and Brennan provide further evidence for their proposed biosynthesis scheme by using an enzyme from M. phlei to specifically incorporate labeled fatty acids into the dimannophosphoinositides. They showed that label from [¹⁴C]palmityl-CoA is incorporated into dimannophosphoinositide C to yield dimannophosphoinositide B. After a short incubation period, this molecule is converted to dimannophosphoinositide A, but with longer incubation periods the product is deacylated to isomeric forms of dimannophosphoinositides B and C.

Brennan continued to work on these glycophosphoinositides after completing his postdoctoral fellowship with Ballou and eventually showed that the lipoglycans (lipomannan (LM) and lipoarabinomannan (LAM)) were multiglycosylated extensions of Ballou's phosphatidylinositol mannosides and are very important in the pathogenesis of tuberculosis and leprosy. More recent research has defined the biochemistry and genetics of synthesis of these molecules. Brennan is currently University Distinguished Professor in the Department of Microbiology, Immunology, and Pathology at Colorado State University. He served on the editorial board of the Journal of Biological Chemistry for several years and was also named Colorado State University Researcher of the Year in 1992.

In 1991, Ballou became Professor Emeritus of Biochemistry at the University of California, Berkeley, although he continued research and teaching for a few years. In recognition of his contributions to science, Ballou has received many awards and honors including election to the National Academy of Sciences (1975), the American Chemical Society's Claude Hudson Award in Carbohydrate Chemistry (1981), the Welch Foundation Lectureship (1972), the University of Notre Dame Reilly Lectureship (1976), the Duke University Belfort Lectureship (1977), a National Science Foundation Senior Fellowship (1961), and a University of California Berkeley Citation (1992). Ballou also served as an editorial board member for the Journal of Biological Chemistry.