From
the Department of Biochemistry and Molecular Biology and Center for
Diabetes Research, Indiana University School of Medicine,
Indianapolis, Indiana 46202
DISCUSSION
We have identified a novel family of
proteins, GNIPs, that interact with glycogenin, a critical component of
the glycogen
biosynthetic pathway. The search for interacting
proteins also selected glycogen synthase and glycogenin, whose ability
to
bind glycogenin was already known.
GNIP1 belongs to
the RBCC subgroup of RING finger proteins, which contain a second zinc
finger known as the B box, followed by a
coiled-coil domain (
26,
27).
COOH-terminal to the RBCC motif, GNIP1 contains a domain similar to the
B30.2-like domain, which was initially identified
as a product of a coding sequence in the
chromosomal region containing the human major histocompatibility complex
class I
(
21). Thus, GNIP1 is a member of the more specific RBCC-B30.2 protein family (
28)
Proteins from this family are involved in various processes including
cell growth and differentiation. This category includes
the following: the RET finger protein, RFP, which
becomes oncogenic when it recombines with the tyrosine kinase domain of
the
RET protooncogene (
29); putative transcription factors, the estrogen-responsive finger protein (
30), EFP, and
Xenopus nuclear factor 7 (
31), Xnf7; the transcriptional regulator Staf-50 (
32); the acid finger protein (
33), AFP; the 52-kDa Sjorgen's syndrome nuclear antigen A (
34), SSA/Ro; and the RING finger B30 protein (
35), RFB30, a protein containing the original B30.2 domain. Recently, several other members of the family have been identified,
including testis RING finger protein (
36), TERF; interferon-responsive RING finger protein 21 (37), RNF21; proteins associated with enterocyte differentiation (
38),
enterophilins. Several proteins with the B30.2-like domain are
associated with pathological conditions. Mutations in the
B30.2 domain of pyrin/marenostrin are thought to
cause the autosomal recessive disease, familial Mediterranean fever (
39,
40). Partial loss of the B30.2 domain in the midline 1 (MID1) protein caused by mutations is responsible for the Opitz G/BBB
syndrome, characterized by developmental midline defects (
41).
Multiple alignment of protein sequences (using the ClustalW algorithm) indicated considerable homology of GNIP1 with the
RBCC-B30.2 proteins, i.e. 36% identity with RFP, 35% identity with testis-abundant finger protein/Ring finger protein 23 (42), 32% identity with TERF,
32% identity with SSA/Ro, and 30% identity with Xnf7.
In attempts to amplify the 5′ end of GNIP
cDNA obtained in two-hybrid screen, we found five distinct extensions.
One would
predict the cDNA for largest isoform of GNIP,
GNIP1. Three distinct cDNA extensions predict three different
5′-noncoding regions
for another protein isoform of GNIP, GNIP2 (Fig.
5). GNIP2 represents the COOH-terminal region of GNIP1, lacking the N-terminal RING finger and the B box domains (Fig.
4). The fifth cDNA extension generates the third isoform of GNIP, GNIP3. This isoform would contain a unique 26-residue sequence
attached to the NH
2 terminus of GNIP2. By searching GenBank
TM with the GNIP1 protein sequence, we identified a
fourth isoform of GNIP, TRIM7, which is identical in sequence to the NH
2-terminal 206-residues of GNIP1 and contains a unique COOH-terminal tail of 15 residues.
All variants are derived from one
region of human chromosome 5, corresponding to theGNIP gene. Characterization of the human
GNIPgene demonstrated a complex gene structure, with multiple differentially spliced transcripts. Some of the variation results
from splicing occurring inside certain exons to produce smaller sub-exons such as 2a, 3a, 3b, and 4a (Fig.
5). The significance of this observation is not clear and could represent the existence of allelic variants.
The existence of multiple products derived from the
GNIPgene is confirmed by other experiments. Northern blot analysis demonstrated transcripts of different sizes in skeletal muscle
and one distinct species in placenta (Fig.
6). Significantly lower levels of GNIP expression were found in heart, brain, and pancreas.
Consistent with our data, other
studies demonstrated that
one of the products of the GNIP gene, TRIM7, is selectively expressed in skeletal muscle in adult mice (
43). However, TRIM7 is ubiquitously distributed in embryonic mouse tissues. Independent evidence for expression of GNIP2 in
skeletal muscle was obtained from sequencing of a mouse EST clone (GenBank
TM accession number
AA517788) from the Barstead myotubes library. This clone contains the entire coding sequence for GNIP2 flanked by 5′-UTR and 3′-UTR,
the latter including the poly(A) tail. Another EST clone (GenBank
TM accession number
AW012184) from mouse kidney might represent a partial sequence of the GNIP1 isoform. Interestingly, both full-length and truncated
versions were found for other members of the RBCC-B30.2 protein family (
37,
43). For example, the gene for RNF21 generates at least three isoforms, due to alternative splicing (
37).
Expression of one specific form in HeLa cells was dramatically
up-regulated by interferon.
It is possible that expression
of individual forms of GNIP in skeletal muscle is
selectively regulated by different stimuli and may be important for
specific
cell functions.
Interaction between isoforms of GNIP and
glycogenin might have important physiological consequences. It is
intriguing to speculate
that GNIP may act to target or sequester glycogenin
to specific cellular locations where glycogen is in demand. The
systematic
study of cellular localization of several members
of RBCC protein family demonstrated that some of these proteins might
target
unique cellular compartments (
43).
To identify other proteins interacting with GNIP, we performed a two-hybrid screen of a human skeletal muscle library using
GNIPt-h as bait. In this screen, we found interaction between GNIPt-h and
desmin.2 We hypothesize that GNIP might target glycogenin to intermediate filaments of the muscle cytoskeleton. However, the biological
significance of this targeting is not clear.
Synthesis of biopolymers is often
regulated at the initiation stage, and so
glycogenin is a candidate to
control glycogen
accumulation. However, our understanding of the
control of glycogenin is still quite limited. In previous work (
44,
45),
we have shown that the ability of purified glycogenin to serve as
substrate for glycogen synthase depends on its glucosylation
state. Identification of protein(s) interacting
with glycogenin leads us to seek regulatory functions. We found that
GNIP2
activates the ability of glycogenin to
self-glucosylate, increasing the
V
max 3–4 times with little or no change in
K
m for UDP-glucose (Fig.
8).
However, GNIP2 does not change the rate of glucosylation of low
molecular weight acceptors indicating that GNIP2 does not
act as regulator of enzymatic activity of
glycogenin. Therefore, the stimulatory effect of GNIP2 on
self-glucosylation presumably
occurs via a change in the conformation of the
glycogenin dimer that creates a better condition for attachment of
glucose
to the existing polysaccharide chain. However, we
cannot exclude the possibility that the activation is secondary to a
more
important targeting role.
Our finding of GNIP in skeletal muscle might help to explain the results obtained by Smythe and colleagues (10).
These authors demonstrated that electrical stimulation of or
epinephrine administration to skeletal muscle caused degradation
of 50% of the glycogen molecules in the muscle
resulting in glycogen-free glycogenin. The subsequent reassociation of
glycogenin
and glycogen synthase was slow and could be
rate-limiting for glycogen re-synthesis in the period of recovery. The
authors
suggested that muscle contains factors that might
be responsible for controlling the rate of reassociation of glycogenin
and
glycogen synthase (10), and we propose that GNIP might be such a factor.
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https://www.sciencedirect.com/science/article/pii/S0003986103006489
GNIP, a Novel Protein That Binds and Activates Glycogenin, the Self-glucosylating Initiator of Glycogen Biosynthesis*
Abstract
Glycogenin
is a self-glucosylating protein that initiates glycogen biosynthesis.
We recently identified a family of proteins, GNIPs, that interact with
glycogenin and stimulate its self-glucosylating activity [J. Biol. Chem.
277 (2002) 19331]. The GNIP gene (also called TRIM7)
encodes at least four distinct isoforms of GNIP, three of which (GNIP1,
GNIP2, and GNIP3) have in common a COOH-terminal B30.2 domain and
predicted coiled-coil regions. Based on Western blot analysis, the GNIP1
protein is widely distributed in tissues. From analysis of a series of
deletion mutants of GNIP2 using the yeast two-hybrid system, the B30.2
domain was found to be responsible for the interaction with glycogenin. A
truncated form of recombinant GNIP2, lacking the NH2-terminal
coiled-coil region, was cross-linked to glycogenin by glutaraldehyde
treatment, supporting the idea that the B30.2 domain was sufficient for
the interaction. In the course of this study, GNIP2 was also found to
interact with itself, via the coiled-coil domain. Heterologous
interactions between GNIP1 and GNIP2 were also detected. Since
glycogenin is also a dimer, higher order multimeric complexes between
glycogenin and GNIPs would be possible.
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