Fig. 1. Secretion Cloning: a novel
approach to isolate secreted proteins.
(A) Overview of the method.
Bacterial colonies from expression
cDNA libraries are picked from agar
plates and individually grown in 96
well blocks. Culture media from 16
clones are pooled and plasmid DNA
prepared by miniprep. Human embryonic
kidney 293T cells are co-transfected
with the cDNA pools and
pAdvantage (pAdv) DNA. After 36
hours, the transfected cells are labeled
and incubated for another 36
hours. Proteins in the cell supernatant
are separated by SDS-PAGE and detected
by autoradiography. Once a candidate
pool is identified, the procedure
is repeated using subpools to individualize
the positive cDNA clone. (B)
Method of sib-selection. Left panel,
bacteria from selected pools were regrown
in a 96 well block and the 16
clones of each pool arrayed in 4x4
wells. The molecular weight of the
candidate protein in each pool is indicated
on the side. Note that pools
grouped next to each other should
differ in their molecular weight. Right
panel, autoradiogram of protein gels.
Each lane is loaded with the supernatant
of cells transfected with a subpool of eight cDNA clones, prepared from one column (letter) or one row (number) of the 96 well block. Note that a
positive clone is identified, when the bands in two lanes have the same molecular weight as the original pool. (C) Example of an SDS-PAGE of radioactive
supernatants of 293T cells transfected with pools of 16 cDNA clones from a Xenopus gastrula library. Note in lanes 12 and 14 bands of 34 and 45 kD,
respectively, which led to the initial identification of secreted Frizzled-related Protein-2 (sFRP2) and Protease inhibitor Nexin-1 (PN-1) in Xenopus. (D)
Optimization of transfection conditions; cDNA clones encoding Protein Phosphatase X (PPX) or Xenopus Cystatin (xCys) were transfected alone (lanes
1-3) or with 10% pAdvantage DNA (pAdv, lanes 4-6). The time between transfection and the addition of 35S-methionine/35S-cysteine is indicated in hours.
Note that the protein yield is highest in the presence of pAdvantage and when metabolic labeling was started 36 hours after transfection. (E) Detection
of Sizzled, PPX and xCys protein in the medium of transfected cells after varying lengths of radioactive labeling. Note that conditioning for 36 hours yields
strong signals of the desired proteins, while longer incubation increases non-specific background.
Fig. 2. Overview of the proteins isolated by secretion cloning. (A)
Positive clones identified in three expression cDNA libraries from early
Xenopus embryos. The number on top of each bar indicates the number of
proteins isolated as bands by SDS-PAGE in the supernatant of transfected
cells. The black portion of the bars indicates unique sequences. (B)
Number of known proteins that have functionally been characterized in
Xenopus, related proteins characterized in other vertebrates but not in
Xenopus and novel proteins not characterized yet. (C) Unique sequences
isolated in the individual expression cDNA libraries. (D) Predicted subcellular
localization of cDNAs related to those of other species or to known
Xenopus cDNAs. Proteins are subdivided into secreted, lysosomal/
endoplasmatic reticulum (ER)/Golgi apparatus and cytoplasmatic/nuclear;
numbers of cDNAs identified are indicated. (E) Subcellular localization of
known and related sequences in each cDNA library. M, maternal library (32-
cell stage); vG, ventral gastrula stage library, dG, dorsal gastrula stage
Fig. 3. xGra1 and xGra2 belong to the
Granulin family of secreted growth
factors. (A) Supernatant culture medium
of human embryonic kidney (293T) cells
transfected with cDNA encoding nonsecreted
green fluorescent protein as
control (GFP), Xenopus Granulin-1 (xGra1)
or Xenopus Granulin-2 (xGra2). Cells
were labeled with 35S-methionine and -
cysteine and their supernatants were
analyzed by SDS-PAGE and autoradiography.
Note that xGra1 is secreted as a
105 kD protein and xGra2 as an 82 kD
protein. (B) Diagrams of xGra1 and xGra2.
The signal peptide cleavage sites are
indicated by triangles. The black boxes
indicate conserved granulin repeats (Pfam
accession number PF00396). The numbers
and guidelines indicate repeats conserved
in both proteins and are based on
sequence similarities. Note that the
granulin repeats 3, 8 and 9 of xGra1 are
missing in xGra2. (C) Signature of the
granulin repeat. Note the conserved spacing
of four cysteine doublets flanked by
two cysteine singletons on each side. (D)
Sequence alignment of xGra1 and xGra2.
xGra1 corresponds to the Granulin provisional
protein sequence previously published
for Xenopus laevis (GenBank accession
number AAH48224) and xGra2 is
novel (GenBank accession number
DQ004683). Identical amino acid residues
are shaded in black and similar or
conserved residues in gray. Dots represent
gaps introduced into the amino acid
sequence in order to obtain optimal alignment.
The signal peptide cleavage site as
predicted by SignalP is indicated with an
arrowhead. Black bars indicate the
granulin repeats and the stars the conserved
cysteine residues. The overall
number of amino acids is indicated at the
end of each sequence. (E-G) Expression
of xGra2 analyzed by whole-mount in
situ hybridization. (E) Four-cell stage
embryo in animal view. Note the high
level of maternal transcripts. (F) Embryo
at late neurula stage in dorsal view showing
spotted expression in the epidermis.
(G) Early tail bud stage embryo in lateral
view. Note expression in the pronephric
Fig. 4. xSOUL is a novel secreted protein of
the SOUL/Heme-binding protein family. (A)
Culture medium of 293T cells transfected with
green fluorescent protein cDNA as control (
GFP) or Xenopus SOUL cDNA (xSOUL ). xSOUL
is secreted as a 23 kD protein. (B) Evolutionary
relationship of xSOUL and other members of
the SOUL/heme binding protein (HBP) family.
Proteins with the greatest sequence similarity
cluster together and branch lengths are proportional
to distance (TreeTop-Phylogenetic Tree
phtree_reduced.html). Only the mature
proteins have been considered. Note that xSOUL
is most closely related to a derived family member
in the plant Arabidopsis thaliana (aSOUL).
The listed proteins have the following GenBank
accession numbers: xSOUL (GenBank accession
number DQ004682); aSOUL (NM101570);
human SOUL (AF117616); mouse SOUL
(AF117614); zebrafish SOUL (BC045936); human
HBP (AF117615); mouse HBP (AF117613);
chick SOUL (AF117612). (C) Comparison of
xSOUL with other SOUL/HBP sequences. Note
that only xSOUL and aSOUL have cleavable
signal peptides (triangle). The putative hemebinding
region as reported by Zylka and Reppert
(1999) is overlined. (D) Four-cell stage embryo
in animal view showing maternal expression of
xSOUL. (E) Anterior view of advanced neurula.
Note weak expression in the anterior brain
(arrowhead). (F) Tailbud stage embryo with
distinct expression in the forebrain, lens, somites
Fig. 5. Xystatin is a novel secreted cysteine proteinase inhibitor. (A) Supernatant
of 293T cells non-transfected (control) or transfected with Xenopus Cystatin cDNA
(xCys ). Note that xCys is secreted as an 18 kDa protein. (B) Phylogenetic tree indicating
the relationship of xCys with other members of the cystatin superfamily. Only the
mature proteins lacking the signal peptide have been aligned. GenBank accession
numbers are as follows: Xystatin (DQ004681); salmon Cystatin (D86628); chick
Cystatin (JO5077); murine Cystatin-C (NM009976); human Cystatin-C (BC013083). (C)
Sequence alignment of Xcys and related members of the cystatin superfamily. Four
conserved cysteine residues known to form two disulphide bonds are labeled with
stars. The conserved active site sequence QxVxG and pro-trp (PW) sequences are
underlined (Brown and Dziegielewska, 1997).
Fig. 6. Whole-mount in situ hybridization of xPN-1, xNuc and xEDJ. Embryos are shown in animal
(A), dorsal (B,D), lateral (C,H,I) or anterior view (E-G). (A-C) Xenopus Proteinase Nexin-1 (xPN-1). (A)
Four-cell stage embryo showing high level of maternal transcripts. (B) Late neurula. Note expression
in the notochord (n) and ear placode (e). (C) Early tail bud stage with additional expression domains in
the lens (le) and ectoderm of the branchial arches (ba). (D-F) Xenopus Nucleobindin (xNuc). (D) Midgastrula
showing expression in the notochord (n). (E) Early neurula with additional signal in the cement
gland (cg). (F) Late neurula. Note strong expression in the cement gland and hatching gland (hg). (G-I)
Xenopus ER-associated DNAJ chaperone (xEDJ). (G) Late neurula with expression in cement gland (cg)
and hatching gland (hg). (H) Late neurula depicting faint signal on the ventral side (arrowhead). (I) Early
tail bud stage. Note expression in the pronephros (pn) and in the hatching gland in the dorsal head.
grn (granulin) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 3, animal view.
grn (granulin) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 20, dorsal view, anterior up.
grn (granulin) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 28, lateral view, anterior left, dorsal up.
hebp2 (heme binding protein 2) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 3, animal view.
hebp2 (heme binding protein 2) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 20, anterior view, dorsal up.
hebp2 (heme binding protein 2) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 30, lateral view, anterior left, dorsal up.
dnajb11 (DnaJ heat shock protein family (Hsp40) member B11) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 18, anterior view, dorsal up.
dnajb11 (DnaJ heat shock protein family (Hsp40) member B11) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 28, lateral view, anterior left, dorsal up.
serpine2 (serpin peptidase inhibitor, clade E (nexin, plasminogen activator inhibitor type 1), member 2) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 3, animal view.
serpine2 (serpin peptidase inhibitor, clade E (nexin, plasminogen activator inhibitor type 1), member 2) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 17, dorsal view, anterior up.
serpine2 (serpin peptidase inhibitor, clade E (nexin, plasminogen activator inhibitor type 1), member 2) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 26, lateral view, anterior left, dorsal up.