The Propeptide Domain of Membrane Type 1-Matrix Metalloproteinase Acts as an Intramolecular Chaperone when Expressed in transwith the Mature Sequence in COS-1 Cells

113

ADAMTS13 belongs to the "a disintegrin and metalloprotease with thrombospondin repeats" family, and cleaves von Willebrand factor multimers into smaller forms. For several related proteases, normal folding and enzymatic latency depend on an NH 2 -terminal propeptide that is removed by proteolytic processing during biosynthesis. However, the ADAMTS13 propeptide is unusually short and poorly conserved, suggesting it may not perform these functions. ADAMTS13 was secreted from transfected HeLa cells with a half-time of 7 h and the rate-limiting step was exported from the endoplasmic reticulum. Deletion of the propeptide did not impair the secretion of active ADAMTS13, indicating that the propeptide is dispensable for folding. Furin was shown to be sufficient for ADAMTS13 propeptide processing in two ways. First, mutation of the furin consensus recognition site prevented propeptide cleavage in HeLa cells and resulted in secretion of pro-ADAMTS13. Second, furin-deficient LoVo cells secreted ADAMTS13 with the propeptide intact, and cotransfection with furin restored propeptide cleavage. In both cell lines, secreted pro-ADAMTS13 had normal proteolytic activity toward von Willebrand factor. In cells coexpressing both AD-AMTS13 and von Willebrand factor, pro-ADAMTS13 cleaved pro-von Willebrand factor intracellularly. Therefore, the ADAMTS13 propeptide is not required for folding or secretion, and does not perform the common function of maintaining enzyme latency.

Section: Discussionmentioning

confidence: 99%

Cleavage of the ADAMTS13 Propeptide Is Not Required for Protease Activity

Majerus

Zheng²,

Tuley

et al. 2003

113

“…The MMP14 propeptide is essential as an intramolecular chaperone, because MMP14 lacking the propeptide is not secreted. However, unlike ADAMTS9, expression of the propeptide in trans restores secretion (27). The propeptides of a number of ADAMs (e.g.…”

Section: Discussionmentioning

confidence: 99%

Regulation of ADAMTS9 Secretion and Enzymatic Activity by Its Propeptide

Koo

Longpré

Somerville

et al. 2007

ADAMTS9 is a secreted, cell-surface-binding metalloprotease that cleaves the proteoglycans versican and aggrecan. Unlike most precursor proteins, the ADAMTS9 zymogen (pro-ADAMTS9) is resistant to intracellular processing. Instead, pro-ADAMTS9 is processed by furin at the cell surface. Here, we investigated the role of the ADAMTS9 propeptide in regulating its secretion and proteolytic activity. Removal of the propeptide abrogated secretion of the ADAMTS9 catalytic domain, and secretion was inefficiently restored by expression of the propeptide in trans. Substitution of Ala for Asn residues within each of three consensus N-linked glycosylation sites in the propeptide abrogated ADAMTS9 secretion. Thus, the propeptide is an intramolecular chaperone whose glycosylation is critical for secretion of the mature enzyme. In addition to two previously identified furin-processing sites (Arg 74 2 and Arg 287 2) the ADAMTS9 propeptide was also furin-processed at Arg 209 . Substitution of Ala for Arg 74 , Arg 209 , and Arg 287 resulted in secretion of an unprocessed zymogen. Unexpectedly, versican incubated with cells expressing this pro-ADAMTS9 was processed to a greater extent than when incubated with cells expressing wild-type, furin-processable ADAMTS9. Moreover, cells and medium treated with the proprotein convertase inhibitor decanoyl-Arg-Val-Lys-Arg-chloromethyl ketone had greater versican-cleaving activity than untreated cells. Following furin processing of pro-ADAMTS9, propeptide fragments maintained a non-covalent association with the catalytic domain. Collectively, these observations suggest that, unlike other metalloproteases, furin processing of the ADAMTS9 propeptide reduces its catalytic activity. Thus, the propeptide is a key functional domain of ADAMTS9, mediating an unusual regulatory mechanism that may have evolved to ensure maximal activity of this protease at the cell surface. ADAMTS4 proteases have critical roles in many biological processes and in inherited and acquired human disorders (1-5). The 19 enzymes of this family share a conserved organization comprising an N-terminal metalloprotease domain and a C-terminal ancillary domain, which contains the thrombospondin type-1 repeats that are the hallmark of the family (6). ADAMTS9 is the largest enzyme of the family, containing 15 thrombospondin type-1 repeats, and its mRNA is widely expressed during embryonic development and in adult tissues (7-9). In previously published work, we showed that when expressed in COS-1 or HEK293F cells, ADAMTS9 is located at the cell surface or within the pericellular matrix (8), suggesting that, despite the lack of a membrane anchor, it could be considered as an operational cell-surface protease. ADAMTS9 can cleave the large aggregating proteoglycans aggrecan and versican (8), suggesting a role in turnover of extracellular matrix. Thus, like its Caenorhabditis elegans ortholog, Gon-1, which is required for cell migration during gonadal morphogenesis (10), it is possible that ADAMTS9 participates in extracellular proteolysi...

“…This is also supported by the fact that SUC and IM possess a comparable number of N-glycans that are distributed similarly over the individual protein domains (7) erone in the context of pro-SI is the first of its kind for a domain that is located at the C-terminal end of a protein. All intramolecular chaperones so far identified in mammalian cells are located immediately at the N-terminal end (33,34,43), presumably to facilitate an early implication in the folding processes of the downstream domains (44). Only two known examples of C-terminally located intramolecular chaperones have been described in bacteria (45) and fungus (43).…”

Section: Expression Of a Full-length Im-mentioning

confidence: 99%

“…A growing family of membrane and secretory proteins contain pro-domains at the N-terminal end that undergo post-translational proteolytic cleavage in the late secretory pathway after the protein has acquired transport competence (26,(33)(34)(35)(36). These domains, called intramolecular chaperones, modulate the folding of this class of proteins directly through interaction with other regions of the protein or by substituting for ER resident chaperones.…”

Section: Expression Of a Full-length Im-mentioning

confidence: 99%

Sucrase Is an Intramolecular Chaperone Located at the C-terminal End of the Sucrase-Isomaltase Enzyme Complex

Jacob¹,

Pürschel²,

Naim³

2002

The sucrase-isomaltase enzyme complex (pro-SI) is a type II integral membrane glycoprotein of the intestinal brush border membrane. Its synthesis commences with the isomaltase (IM) subunit and ends with sucrase (SUC). Both domains reveal striking structural similarities, suggesting a pseudo-dimeric assembly of a correctly folded and an enzymatically active pro-SI. The impact of each domain on the folding and function of pro-SI has been analyzed by individual expression and coexpression of the individual subunits. SUC acquires correct folding, enzymatic activity and transport competence and is secreted into the external milieu independent of the presence of IM. By contrast, IM persists as a mannose-rich polypeptide that interacts with the endoplasmic reticulum resident molecular chaperone calnexin. This interaction is disrupted when SUC is coexpressed with IM, indicating that SUC competes with calnexin for binding of IM. The interaction between SUC and the membrane-anchored IM leads to maturation of IM and blocks the secretion of SUC into the external milieu. We conclude that SUC plays a role as an intramolecular chaperone in the context of the pro-SI protein.To our knowledge all intramolecular chaperones so far identified are located at the N-terminal end. SUC is therefore the first C-terminally located intramolecular chaperone in mammalian cells.Membrane and secretory proteins are subject to a complex array of cotranslational and post-translational processes that precede sorting to the organelle in which they exert their biological functions. The most crucial events take place in the ER 1 and include folding, subunit assembly, and in many cases oligomerization (for review see Ref. 1). These steps, individually or altogether, lead ultimately to the acquisition of the protein to a transport-competent conformation that enables its egress from the ER. A number of resident proteins of the ER, such as the molecular chaperones BiP and calnexin and protein disulfide isomerase, play primordial roles in these processes that vary from binding of unfolded proteins to the formation of disulfide bonds between individual subunits or within a single polypeptide chain (2-4). A large number of proteins assemble into homodimers, oligomers, or heterodimers preceding protein exit from the ER (5, 6). Correct protein folding of the individual monomers or subunits is required for optimal completion of these events. Many proteins are known not to go into dimers or heterodimers, such as the brush border proteins sucrase-isomaltase (SI) (7), angiotensin-converting enzyme (8), and maltase-glucoamylase (9). An interesting and common feature of these proteins is their composition of an even number of homologous domains. SI, for example, is an enzyme complex that is made of two subunits that share Ն 35% of amino acid sequence similarity and 41% of conserved sequences (10). Although no three-dimensional structure of this large protein is so far available, algorithmic predictions suggest that the two large domains are folded similarly and that...