The Nerve Hemoglobin of the Bivalve Mollusc Spisula solidissima: MOLECULAR CLONING, LIGAND BINDING STUDIES, AND PHYLOGENETIC ANALYSIS

Sylvia Dewilde,Bettina Ebner,Evi Vinck,Kambiz Gilany,Thomas Hankeln,Thorsten Burmester,Jill Kreiling,Carol Reinisch,Jacques R Vanfleteren,Laurent Kiger,Michael C Marden,Christian Hundahl,Angela Fago,Sabine Van Doorslaer,Luc Moens

doi:10.1074/jbc.m509486200

Abstract

Members of the hemoglobin (Hb) superfamily are present in nerve tissue of several vertebrate and invertebrate species. In vertebrates they display hexacoordinate heme iron atoms and are typically expressed at low levels (microM). Their function is still a matter of debate. In invertebrates they have a hexa- or pentacoordinate heme iron, are mostly expressed at high levels (mM), and have been suggested to have a myoglobin-like function. The native Hb of the surf clam, Spisula solidissima, composed of 162 amino acids, does not show specific deviations from the globin templates. UV-visible and resonance Raman spectroscopy demonstrate a hexacoordinate heme iron. Based on the sequence analogy, the histidine E7 is proposed as a sixth ligand. Kinetic and equilibrium measurements show a moderate oxygen affinity (P(50) approximately 0.6 torr) and no cooperativity. The histidine binding affinity is 100-fold lower than in neuroglobin. Phylogenetic analysis demonstrates a clustering of the S. solidissima nerve Hb with mollusc Hbs and myoglobins, but not with the vertebrate neuroglobins. We conclude that invertebrate nerve Hbs expressed at high levels are, despite the hexacoordinate nature of their heme iron, not essentially different from other intracellular Hbs. They most likely fulfill a myoglobin-like function and enhance oxygen supply to the neurons.

Highlights

Members of the hemoglobin (Hb) superfamily are present in nerve tissue of several vertebrate and invertebrate species
In the gastropod mollusc Aplysia depilans, the oxygenation state of the nerve hemoglobins” (nHbs) was correlated with the electrical activity of the neural ganglia, and firing was shown to be proportional to the degree of oxygenation of the globin (8)
The nHbs appear to be expressed in the glial cells surrounding neurons, as reported for the clams T. alternata and Spisula solidissima (10), the water-dwelling gastropods Lymnaea stagnalis and Planorbis corneus (11), the nemertean C. lacteus (7), and the annelid A. aculeata (12)

Summary

The Nerve Hemoglobin of the Bivalve Mollusc Spisula solidissima

This results in complex ligand binding kinetics, of which the functional implications still have to be revealed (15, 16) Several of these features (e.g. hexacoordination, moderate oxygen affinity) of invertebrate nHbs are shared with the neuroglobins (Ngb), which have been identified in many vertebrate taxa, including mammals, birds, amphibians, and fishes (17). For further understanding of both vertebrate Ngb and invertebrate nHb functions, we have aimed at the molecular characterization of the nHb of the Atlantic surf clam S. solidissima (14, 28, 29) This protein is an example of a hexacoordinate nHb, which is abundantly expressed in glial cells and lends the nerves of this organism a bright red appearance

MATERIALS AND METHODS

RESULTS AND DISCUSSION

Horse Mb