Malectin is an ubiquitously expressed protein

Malectin expression was analysed on the RNA level by WMISH and semiquantitative RT-PCR as well as on the protein level by immunodetection. By WMISH, stage 1 to stage 13 embryos showed a weak and diffuse staining that did not remarkably differ from unspecific background staining (not shown). Region specific expression of malectin transcripts was first observed at neurula stage 1 (stage 18 to 20) in the anterior neural ectoderm, the neural folds and the neural crest (figure 3.3.2 A; stage 18 and 20). At tadpole stage 32, malectin was broadly expressed including the neural crest placodes, otic vesicle, pronephros, the tail tip, the neural tube but also in the retina. Interestingly, at this stage malectin expression was upregulated in a distinct structure in the mid-dorsal head region, namely the hatching

Figure 3.3.1 Sequence comparison of Xenopus laevis malectin with eukaryotic homologues. Ali��ne�

eukaryoti�� protein sequen��es an� ��orrespon�in�� a����ession number are state� to the left. Small numbers to the ri��ht in�i��ate the amino a��i� position. Resi�ues �ifferin�� from a ��onsensus sequen��e are hi��hli��ht-e� in rhi��hli��ht-e�. Protein fra��ment of Xenopus laevis male��tin whi��h was use� for stru��tural analysis by N�R is marke� by a blue bar �S��hallus et al., ��00��. Sequen��e analysis usin�� S�ART-tool pre�i��te� putative protein

�omains: an N-terminal si��nal pepti�e �aa 1- ��5; re� bar�, an interme�iate pre�i��te� ��lobular �omain �aa

��6- ��56, blue bar�, an� an hy�rophobi�� C-terminal transmembran �omain �aa ��56- ��76, yellow bar�. Note, that male��tin sequen��e ��ontains a ne��ative ��har��e� serial sequen��e of ��lutami�� a��i� �E� at aa ��30- ��4��.

gland (figure 3.3.2; A 32, inset). Malectin remained expressed in neural crest derivativesfigure 3.3.2; A 32, inset). Malectin remained expressed in neural crest derivatives32, inset). Malectin remained expressed in neural crest derivatives during subsequent development, while additional transcripts appeared in the differentia-ting pancreas at stage 41.

To determine the exact time point of malectin transcription, the original clone obtained from the cDNA library screen (pBK-CMV-p150) was used to design malectin specific oli-gonucleotides to analyse temporal malectin gene expression by semiquantitative RT-PCR (figure 3.3.2, B). In contrast to the WMISH, semiquantitative RT-PCR on total RNA ex-tracts from stage V oocytes and different embryonic stages embryos (stages V, 0- 42) re-vealed a presence of malectin transcripts already in oogenesis that persisted in at constant expression level throughout development.

In order to detect the time course of protein synthesis, a malectin specific antibody was generated and used for immunodetection analysis of the malectin protein in different em-bryonic stages (figure 3.3.2, B). According to the detailed sequence information obtained by the collaborating group of Claudia Muhle-Goll (SMART protein domoin prediction tool), it was suggested that malectin was a type I transmembrane protein (Schallus et al., 2008). For this reason, membrane protein fractions of embryos with different stages were analysed for the presence of malectin protein. Embryos of different developmental stages were homogenised, and membrane debries pelleted. The malectin protein was specifically detected using a newly generated Xenopus laevis specific malectin antibody that was di-rected against the intermediate lectin like domain of the protein (aa 30-254, see 2.2.10).

This antibody detected very specifically one single protein band with an estimated size of 30kD that matched the predicted size of malectin protein (30kD). This protein band was evident in all stages tested. Hence, it was demonstrated that also malectin protein was present from early embryogenesis onwards indicating a constant translational activity.

Expression analysis of malectin in adult tissues of Xenopus laevis revealed an ubiquitous expression in all analysed tissues albeit with varying transcription levels (figure 3.3.2; C).

Weak expression was detected in eye, kidney and the heart whereas muscle, gall bladder, stomach, liver and lung. Also the pancreas demonstrated high transcript levels. According to the specific expression of XPDIp that was detected in the pancreas and stomach tissue preparations (Afelik et al., 2004) it was excluded that the broad expression derived from cross contamination during the RNA purification procedure.

Altogether, malectin expression was shown to be present throughout development in a rather ubiquituosly manner than in a germlayer specific way or in a spatially restricted territory.

Figure 3.3.2: Spatial and temporal expression profile of malectin in Xenopus laevis embryos and adult.

(A) Spatial �istribution of male��tin was analyse� by W�ISH. pBK-C�V-p150 full len��th ��lone was linearise�

with BamHI an� �i��-labele� antisense RNA was trans��ribe� with T7-RNA-polymerase. From sta��e 1� on-war�s, male��tin is expresse� in the anterior neuroe��to�erm �ne� an� neural ��rest �n��, white arrow hea�s�.

At sta��e 3��, it is restri��te� to the retina �re�, oti�� vesi��le �ot�, epibran��hial pla��o�es �eb�, pronephros �pn�

an� the tail tip �tp� as well as to the hat��hin�� ��lan� �h��, front view of the hea�, inset�. At sta��e 41, tran-s��ripts are �ete��te� in the liver �li, frame� in re��, the a�ja��ent ventral pan��reas �vp, frame� in white� an�

�orsal pan��reas ��p� as well as in bran��hial ar��hes �ba�, oesopha��us �oe� an� pro��to�eum �p��. (B) A��tiva-tion of male��tin ��ene expression was �etermine� by semiquantitative RT-PCR on total RNA extra��ts an���ene expression was �etermine� by semiquantitative RT-PCR on total RNA extra��ts an�

by Westernbot on embryoni�� protein extra��ts. Total RNA amount was equalise� to 100 n����µl for 10µl rea��-tion for ���NA synthesis. �H_��� was use� as ne��ative ��ontrol. Histone H4 was use� equalise RNA amounts.

�li��onu��leoti�es use� were p150��male��tin-RT_for�� p150��male��tin-RT rev, Histone H4-for�� Histone H4 rev;

�P�Ip-for��rev. �ale��tin trans��ription is persistent throu��hout �evelopment as ��ompare� to pan��reas

spe-��ifi�� protein �isulfi�e isomerase ��P�Ip� that is initiate� at sta��e 39. �evelopmental sta��es �Nieuwkoop an� Faber, 1967� are in�i��ate� as numbers on top of ea��h lane. VI=sta��e VI oo��yte. For immuno�ete��tionVI=sta��e VI oo��yte. For immuno�ete��tionFor immuno�ete��tion analysis, membrane protein fra��tions were ��enerate� of �ifferent �evelopmental sta��es. 10 embryos wereof �ifferent �evelopmental sta��es. 10 embryos were homo��enise� usin�� a syrin��e. The suspension was ��entrifu��e� to obtain membraneous ��ompartments whi��h were resolve� in ��0 µl sample buffer ��embryo. 10µl sample �0.5 embryo equivalents� were loa�e�

per lane of a 1��.5% S�S-�el. After S�S-PA�E, proteins were transferre� onto a nitro��ellulose membrane.

En�o��enous male��tin protein was �ete��te� usin�� a poly��lonal male��tin spe��ifi�� antibo�y �rabbit-anti-male��tin protein was �ete��te� usin�� a poly��lonal male��tin spe��ifi�� antibo�y �rabbit-anti-male��tin; 1:15000� an� the se��on�ary antibo�y ��oat-anti-rabbit-HRP �Santa Curz, 1:5000�. Numbers on top of the lanes in�i��ate� �evelopmental sta��es; BL = ba��terila lysate was use� as ne��ative ��ontrol, T�4=

purifie� male��tin protein �aa 30-��0�� whi��h was use� as anti��en for rabbit immunisation. The ban� shift in sta��e 0 results from a ��el artefa��t (C) �ale��tin expression in a�ult tissues was anaylse� by RT-PCR. It shows ubiquitiuos �istribution in ��omparison to �P�Ip whi��h is �ete��te� only in pan��reas an� stoma��h.only in pan��reas an� stoma��h.in pan��reas an� stoma��h.

Abbreviations are not �efine� in A: ��all bla��er ���b�; heart �he�; intestine �in�; ki�ney �ki�; lun�� �lu�; mus��leare not �efine� in A: ��all bla��er ���b�; heart �he�; intestine �in�; ki�ney �ki�; lun�� �lu�; mus��lenot �efine� in A: ��all bla��er ���b�; heart �he�; intestine �in�; ki�ney �ki�; lun�� �lu�; mus��le

The presence of malectin from the beginning of embryogenesis on to the mature amphi-bian, together with its dispersed transcript localisation and late onset of expression in thelocalisation and late onset of expression in theand late onset of expression in the pancreatic tissue, favored the idea that malectin rather maintained a general function du-maintained a general function du-general function du-ring embryogenesis than a restricted role dudu-ring pancreas organogenesis.