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J Cell Biol
1993 Feb 01;1204:1059-67. doi: 10.1083/jcb.120.4.1059.
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Identification and characterization of thrombospondin-4, a new member of the thrombospondin gene family.
Lawler J
,
Duquette M
,
Whittaker CA
,
Adams JC
,
McHenry K
,
DeSimone DW
.
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A new member of the thrombospondin gene family, designated thrombospondin-4, has been identified in the Xenopus laevis genome. The predicted amino acid sequence indicates that the protein is similar to the other members of this gene family in the structure of the type 3 repeats and the COOH-terminal domain. Thrombospondin-4 contains four type 2 repeats and lacks the type 1 repeats that are found in thrombospondin-1 and 2. The amino-terminal domain of thrombospondin-4 has no significant homology with the other members of the thrombospondin gene family or with other proteins in the database. RNAse protection analysis establishes that the initial expression of Xenopus thrombospondin-4 is observed during neurulation. Levels of mRNA expression increase twofold during tailbud stages but decrease by the feeding tadpole stage. The size of the thrombospondin-4 message is 3.3 Kb and 3.4 Kb in the frog and human, respectively. Northern blot analysis of human tissues reveals high levels of thrombospondin-4 expression in heart and skeletal muscle, low levels in brain, lung and pancreas and undetectable levels in the placenta, liver and kidney. These data establish the existence of a new member of the thrombospondin gene family that may participate in the genesis and function of cardiac and skeletal muscle.
Figure 1. Alignment of Xenopus thrombospondin-4 clones. Restriction
endonuclease sites are indicated for the two families (TSP-4A
and TSP-4B). The clones that have been isolated in the first (XF1-
XF4), second (XSS-XS10), and third (XTI1-XTI4) rounds of
screening have been grouped into their appropriate family by restriction
endonuclease mapping and nucleotide sequencing.
lqgure 2. Northern blot of Xenopus stage
17 RNA. Two micrograms of total stage
17 mRNA was electrophoresed and blotted.
The blot was probed with the XF3
clone. The positions and sizes of the
markers are indicated on the letL
Figure 3. The nucleotide sequence and corresponding amino acid sequence of Xenopus thrombospondin-4. The predicted site for signal
sequence cleavage is indicated (open triangle). The termination codon TAA and the polyadanylation signal AATAAA are underlined. The
sites for potential N-linked glyeosylation are circled and the site for potential ~-hydroxylation is boxed. Note that asparagine(314) could
be either glycosylated or ~-l~xlmxylated. These sequence data are available from EMBL/GenBank/DDBJ under accession number, Z19091.
Figure 4. The amino acid sequence of the type 2 (A) and 3 (B)
repeats of thrombospondin-4. The amino acid sequences for the
four type 2 repeats P(217)-K(394) and seven type 3 repeats
(D[416]-I[647]) have been aligued. The positions of gaps ~
marked by dashes. The consensus sequence for the type 3 repeats
of Xenop~ thmmbospondin-4 is compared to that for human and
mouse thrombospondin-1 and chicken thrombospondin-2 at the
bottom of the figure. The underline indicates that an N occupies
one of the positions that is occupied by a D.
Figure 5. Developmental expression of thrombospondin-4 mRNAs
during Xenopus development as determined by RNase protection
analyses. Ten embryo equivalents total mRNA per lane were bybridized
with a 3~P-labeled thrombospondin-4 antisense transcript
synthesized in vitro. EFI-,~ mRNA analysis is included as a control
for RNA loading. The relative stage-specific levels of EFI-,v shown
here are equivalent to those described by Krieg et al. (1989). Embryos
were staged according to Nieuwkoop and Faber (1967). Stage
8 corresponds to the mid-blastula, which is marked by the onset
of zygotic transcription in Xenopus (Newport and Kirschner, 1982).
(P), probe alone, unhybridized transcript, no RNAse; (t) tRNA
control lane, no protected fragments after RNAse digestion. Stages
include: (1) fertilized egg, (8) mid-blastula, 00-12) early-late gastrula,
07) neurula, (25-35) tailbud stages, (45) feeding tadpole.
Thrombospondin-4 protected fragment is ,,o300 nucleotides. EFI-a
protected fragment is ,x,75 nucleotides. Full length EFI-c~ probe (90
nucleotides) is not shown.
Figure 6. The expression of thrombospondin-4 in adult human tissue.
A northern blot ofpoly A + RNA from adult human heart (a),
brain (b), placenta (c), lung (d), liver (e), skeletal muscle (f), kidney
(g), and pancreas (h). The blot was probed with a 2.2-kb fragment
of Xenopus thrombospondin-4. The positions and sizes (kb)
of the markers are indicated on the left.
Figure 7. Schematic model of thrombospondin-4. The molecular
architecture of thrombospondin-4 is shown below that of
thrombospondin-1 or 2. The NH2 terminal is to the left and the
COOH terminal is to the right. Whereas the NH2-terminal domains
are similar in size, they share very little sequence homology.
Two interchain disulfides of thrombospondin-1 and 2 are depicted
as vertical lines between the NH~-terminal domain and the region
of homology with procollagen. Whereas two cysteine residues are
present in a similar location in thrombospondin-4, the structure of
the protein that contains the thrombospondin-4 polypeptide remains
to be determined.
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