SBNet - Research Reports 1999

Xiao-Dong Su

Position: "Forskare" (LU)

Project: Crystallography at MAX II

Part of my job (20%-30%) is user support for the beam line BL711 at MAX lab. Besides the user support, I have also been taking part in teaching:

(1) Advisor (handledare) of two project students (examensarbetare):

Rosmarie Friemann has finished her Master thesis entitled "over-expression and purification of immunoglobulin like domains of Drosophila melanogaster neuroglian" with me early 1999, and she is now a Ph.D. student in the department of molecular biology, BMC, Uppsala.

Fredrik Berglöf is still doing his thesis work in our lab, expecting to finish early 2000.

(2) Tutor of a graduate course in "protein crystallography";

(3) Lab designer and instructor of KEM103 (Physical Chemistry).

My own research project :

Structure and function studies of cell adhesion molecule L1

Cell adhesion molecules (CAMs) play important roles in many diseases including cancer metastatic behaviour, one of such player is neural CAM L1 which mediates homophilic and heterophilic adhesion events not only in nervous systems but also in leukocytes and some epithelial cells. It has been shown that the expression of L1-CAM inversely correlates with the metastatic capacity of a lymphoma cell line in mice, therefore, to understand this mechanism may provide valuable clues for cancer research and therapy. The aim of this research project is to determine the crystal structures of the functional parts that mediate cell adhesion of L1-CAMs by x-ray crystallography. We have over-expressed, purified the first 4 domains of Drosophila neuroglian (L1 in insect) that has been shown to be important to mediate homophilic cell adhesion in Drosophila S2 cells. The over-expression of other domains containing the RGD integrin-binding site of mammalian L1-CAM is also under construction.

Nrg-4D: It is the first four N-terminal Ig-like domains of neuroglian, a Drosophila L1-CAM that shares 38% sequence identity with hemolin. It contains three N-linked glycosylation sites; and Mass spectroscopy data showed that the recombinant Nrg-4D expressed in High 5 cells is glycosylated non-homogeneously, with 5-6 KD molecular weight difference from the calculated non-glycosylated value. This work has started since my post-doc in Caltech.

The protein was over-expressed using a high titre virus stock in either Sf-9 or High 5 cells. The production level was about 2-3 mgs per litre of culture medium. The Nrg-4D has been purified in reasonable amount here in Lund University. In addition to the ultracentrifugation results published on Science 281 991-995, (1998), more biochemical analysis and crystallisation trials have been tried, and micro-crystals of Nrg-4D have been observed. A manuscript is in preparation for this work.

Over-expression and refolding of neuroglian Ig domain fragments in E. coli:

Parallel to the baculovirus expression system, E. coli expression is also under study to see if we can refold in vitro the non-glycosylated neuroglian fragments. The final goals are to obtain both glycosylated and non-glycosylated structure of neuroglian fragments to compare the structural and functional differences. Part of this work has been carried out by a project student, so far she has got good level of inclusion body over-expression of the first six Ig domains as 3 times two domains (d1d2, d3d4, d5d6) in E. coli using the pET28a vector. So far, the Nrg-d5d6 has been successfully refolded according to results obtained from gel filtration chromatography, native gel electrophoresis and CD spectroscopy, and small crystals have been obtained. A manuscript is in preparation for this part of work.

Human L1: This protein is responsible for a series of inherited neural disorders and some type of cancer metastasis. Cancerfonden has supported me to determine the structure of this family of neural CAMs.

The over expression of the different domains of human L1 CAM has also been started and will be continued from baculovirus and E. coli expression systems.

Financial situation:

My salary and some running costs have been supported by SSF via SBNet. I have obtained financial support from:

1) 200, 000 SEK each year for two years (1999 and 2000) from Cancerfonden;

2) About 240,000 SEK for equipment from Crafoordska stiftelsen;

3) 60,000 SEK for equipment from Kungl. Fysiografiska Sällskapet.

Invited lectures:

1) The crystal structure of horseshoe shaped hemolin monomer: a novel mechanism for homophilic adhesion; Feb. 16, 1999, at Dept. of Genetics (host: Ingrid Faye) and Dept of Biochemistry (host: Per Nordlund), Stockholm Univ.

2) The crystal structure determination of an insect LPS (lipopolysaccharide) binding protein: hemolin; April 22, 1999, at the Graduate school of drug research, royal Danish school of pharmacy.

True collaborations in the health care sector:

Dr. Magnus Abrahamson's group at the Dept. of Clinical Chemistry, University of Lund, University Hospital, Lund, Sweden.

Dr. Bruno Villoutreix at Department of laboratory Medicine, Division of Clinical Chemistry, University Hospital, Malmö, Sweden

Visiting foreign post-doc:

Dr. Sun, Xin (Ph.D. CMB, Karolinska Inst. 1998), during April to Nov. 1999

SBNet Latest update at 3 April, 2000.