Jan Sedzik

The three-dimensional structure of P2 myelin protein

The three‐dimensional structure of P2 protein from peripheral nervous system myelin has been determined at 2.7 A resolution by X‐ray crystallography. The single isomorphous replacement/anomalous map was interpreted using skeletonized electron density on a computer graphics system. An atomic model was built using fragment fitting. The structure forms a compact 10‐stranded up‐and‐down beta‐barrel which encapsulates residual electron density that we interpret as a fatty acid molecule. This beta‐barrel shows some similarity to, but is different from, the retinol binding protein family of structures. The relationship of the P2 structure to a family of cytoplasmic, lipid binding proteins is described.

Bovine P2 myelin basic protein crystallizes in three different forms

Abstract: P2 protein is a minor component of the myelin membrane. We have crystallized this protein for high‐resolution crystallographic study. Three crystal morphologies are available. Two of them are from ammonium sulfate, and one is from polyethyleneglycol (PEG). The unit cell of the most suitable crystals from PEG 4000 has the dimensions a= 91.3 Å, b= 99.8 Å, c= 56.0 Å; is of space group P212121; and contains up to four molecules per asymmetric unit. The limit of resolution is 2.7 Å.

Glycans of myelin proteins

Human P0 is the main myelin glycoprotein of the peripheral nervous system. It can bind six different glycans, all linked to Asn93, the unique glycosylation site. Other myelin glycoproteins, also with a single glycosylation site (PMP22 at Asn36, MOG at Asn31), bind only one glycan. The MAG has 10 glycosylation sites; the glycoprotein OMgp has 11 glycosylation sites. Aside from P0, no comprehensive data are available on other myelin glycoproteins. Here we review and analyze all published data on the physicochemical structure of the glycans linked to P0, PMP22, MOG, and MAG. Most data concern bovine P0, whose glycan moieties have an MW ranging from 1,294.56 Da (GP3) to 2,279.94 Da (GP5). The pI of glycosylated P0 protein varies from pH 9.32 to 9.46. The most charged glycan is MS2 containing three sulfate groups and one glucuronic acid; whereas the least charged one is the BA2 residue. All glycans contain one fucose and one galactose. The most mannose rich are the glycans MS2 and GP4, each of them has four mannoses; OPPE1 contains five N‐acetylglucosamines and one sulfated glucuronic acid; GP4 contains one sialic acid. Furthermore, human P0 variants causing both gain and loss of glycosylation have been described and cause peripheral neuropathies with variable clinical severity. In particular, the substitution T95→M is a very common in Europe and is associated with a late‐onset axonal neuropathy. Although peripheral myelin is made up largely of glycoproteins, mutations altering glycosylation have been described only in P0. This attractive avenue of research requires further study.

High-resolution structural model of porcine P2 myelin membrane protein with associated fatty acid ligand: Fact or artifact?

Myelin membrane is a biological complex of glial cells origin; it is composed of 25% (w/w) proteins and 75% lipids, and more than 300 proteins are associated with central nervous system myelin (for peripheral nervous system myelin, such data are lacking). Myelin plays an important role in maintaining propagation of nerve signals. To uncover the nature of propagation phenomena, it is essential to study biochemistry of myelin proteins and lipids, myelin composition, and myelin structure. Nearly all myelin proteins are like antigens, causing clinically well‐defined devastating diseases; multiple sclerosis and Guillain‐Barré syndrome are two of them. In this article, a high‐resolution study (1.8 Å) of porcine myelin P2 protein is presented. Myelin was purified from porcine intradural spinal roots, which were stored at −80°C for 10 years before myelin and P2 protein were purified (spinal roots were a gift of Prof. Kunio Kitamura, Saitama Medical School). The three‐dimensional structural analysis uncovered embedded 18‐carbons‐long fatty acid. Some speculative interpretation is presented, to uncover how this ligand of fatty acid may form cholesterol ester and stabilize the myelin structure or form simple raft microdomain. Protein crystallography indicates that the ligand may be 18‐carbons‐long fatty acid. This is unlike previous work with mass spectrometry, in which three ligands were determined. In other protein crystallography‐based studies of P2 (bovine), an oleic fatty acid was suggested, but, for recombinant (human) protein, palmitic acid was found. There is no fatty acid ligand in equine P2 protein.

Myelin basic protein purified on an ion-exchange continuous polymer bed in the presence of ethylene glycol and salt possesses activity against p-nitrophenyl acetate.

In this paper we describe a fast and mild method based on the use of a unique cation exchanger and buffers containing ethylene glycol and salt for the purification of the myelin basic protein (MBP; MW 18.5 kDa). MBP thus purified hydrolyses catalytically p-nitrophenyl acetate. This esterase activity facilitates not only the purification of MBP but also indicates that probably it is in its native state, i.e. there is a good chance that the purified molecules are structurally and chemically identical. This is a prerequisite to obtain crystals appropriate for x-ray diffraction and other studies.

Ups and downs of protein crystallization: studies of protein crystals by high-performance capillary electrophoresis.

High-performance capillary electrophoresis is a high-technology micro-separation method. Short run time, full automation and minute amounts of sample make it a very attractive technique. In this report we describe studies of protein crystals by capillary electrophoresis. We show how high-performance capillary electrophoresis can be used effectively for rapid evaluation and examination of the protein solution used for crystallization, the protein crystals (solubilized) and surrounding mother liquor. With coated capillaries, the runs were reproducible and disturbing effects, such as electroendosmosis and interaction of the proteins with the capillary wall, were suppressed efficiently. We recommend this new technique as a powerful and routine companion to protein crystallography.

Sequence motifs of myelin membrane proteins : Towards the molecular basis of diseases

The shortest sequence of amino acids in protein containing functional and structural information is a “motif.” To understand myelin protein functions, we intensively searched for motifs that can be found in myelin proteins. Some myelin proteins had several different motifs or repetition of the same motif. The most abundant motif found among myelin proteins was a myristoylation motif. Bovine MAG held 11 myristoylation motifs and human myelin basic protein held as many as eight such motifs. PMP22 had the fewest myristoylation motifs, which was only one; rat PMP22 contained no such motifs. Cholesterol recognition/interaction amino‐acid consensus (CRAC) motif was not found in myelin basic protein. P2 protein of different species contained only one CRAC motif, except for P2 of horse, which had no such motifs. MAG, MOG, and P0 were very rich in CRAC, three to eight motifs per protein. The analysis of motifs in myelin proteins is expected to provide structural insight and refinement of predicted 3D models for which structures are as yet unknown. Analysis of motifs in mutant proteins associated with neurological diseases uncovered that some motifs disappeared in P0 with mutation found in neurological diseases. There are 2,500 motifs deposited in a databank, but 21 were found in myelin proteins, which is only 1% of the total known motifs. There was great variability in the number of motifs among proteins from different species. The appearance or disappearance of protein motifs after gaining point mutation in the protein related to neurological diseases was very interesting.

Crystallization of P2 myelin protein

Single crystals of bovine P2 myelin protein have been grown in polyethylene glycol 4000 by the hanging-drop vapor diffusion method. Crystals belonging to space group P2(1)2(1)2(1) with cell dimensions a = 91.8 A, b = 99.5 A, c = 56.5 A (1 A = 0.1 nm). The diffraction pattern extends to better than 2.3 A resolution.