Structural analysis and tissue localization of human C4.4A: a protein homologue of the urokinase receptor

TY - JOUR

T1 - Structural analysis and tissue localization of human C4.4A

T2 - a protein homologue of the urokinase receptor

AU - Hansen, Line V.

AU - Gårdsvoll, Henrik

AU - Nielsen, Boye S

AU - Lund, Leif R

AU - Danø, Keld

AU - Jensen, Ole N.

AU - Ploug, Michael

PY - 2004/6/15

Y1 - 2004/6/15

N2 - C4.4A, a structural homologue of the urokinase-type plasminogen activator receptor (uPAR), was originally identified as a metastasis-associated membrane protein, but little is known about its structural and functional properties. Therefore, we expressed, purified and characterized a soluble truncated form of human C4.4A, and used this protein to produce specific polyclonal anti-C4.4A antibodies. By immunohistochemistry we observed a pronounced surface staining for C4.4A in suprabasal keratinocytes of chronic human wounds and found C4.4A expression markedly upregulated in migrating keratinocytes during re-epithelisation of incisional skin wounds. Phorbol-ester-induced hyperplasia of mouse skin is also accompanied by a significant induction of C4.4A expression in the multilayered, suprabasal keratinocytes. C4.4A contains two Ly-6 (leucocyte antigen 6)/uPAR/alpha-neurotoxin modules. Our recombinant human C4.4A is extensively modified by post-translational glycosylation, which include 5-6 N-linked carbohydrates primarily located in or close to its second Ly-6/uPAR/alpha-neurotoxin module and approximately 15 O-linked carbohydrates clustered in a Ser/Thr/Pro-rich region at the C-terminus. A highly protease-sensitive region (Tyr200-Arg204) is located between these two clusters of N- and O-linked carbohydrates. The natural, glycolipid-anchored C4.4A from amnion membranes of human term placenta exhibits similar properties. Using recombinant, soluble C4.4A or MCF 7 cells, which express significant amounts of GPI-anchored C4.4A, we find no evidence for an interaction between C4.4A and uPA, a property suggested previously for rat C4.4A. Collectively these data indicate that C4.4A, although being a structural homologue of uPAR, is unlikely to have a functional overlap with uPAR.

AB - C4.4A, a structural homologue of the urokinase-type plasminogen activator receptor (uPAR), was originally identified as a metastasis-associated membrane protein, but little is known about its structural and functional properties. Therefore, we expressed, purified and characterized a soluble truncated form of human C4.4A, and used this protein to produce specific polyclonal anti-C4.4A antibodies. By immunohistochemistry we observed a pronounced surface staining for C4.4A in suprabasal keratinocytes of chronic human wounds and found C4.4A expression markedly upregulated in migrating keratinocytes during re-epithelisation of incisional skin wounds. Phorbol-ester-induced hyperplasia of mouse skin is also accompanied by a significant induction of C4.4A expression in the multilayered, suprabasal keratinocytes. C4.4A contains two Ly-6 (leucocyte antigen 6)/uPAR/alpha-neurotoxin modules. Our recombinant human C4.4A is extensively modified by post-translational glycosylation, which include 5-6 N-linked carbohydrates primarily located in or close to its second Ly-6/uPAR/alpha-neurotoxin module and approximately 15 O-linked carbohydrates clustered in a Ser/Thr/Pro-rich region at the C-terminus. A highly protease-sensitive region (Tyr200-Arg204) is located between these two clusters of N- and O-linked carbohydrates. The natural, glycolipid-anchored C4.4A from amnion membranes of human term placenta exhibits similar properties. Using recombinant, soluble C4.4A or MCF 7 cells, which express significant amounts of GPI-anchored C4.4A, we find no evidence for an interaction between C4.4A and uPA, a property suggested previously for rat C4.4A. Collectively these data indicate that C4.4A, although being a structural homologue of uPAR, is unlikely to have a functional overlap with uPAR.

KW - Amino Acid Sequence

KW - Animals

KW - Breast Neoplasms

KW - Cell Adhesion Molecules

KW - Dermatologic Surgical Procedures

KW - GPI-Linked Proteins

KW - Humans

KW - Hyperplasia

KW - Immunohistochemistry

KW - Mice

KW - Mice, Inbred C57BL

KW - Molecular Sequence Data

KW - Peptide Fragments

KW - Peptide Mapping

KW - Placenta

KW - Protein Interaction Mapping

KW - Protein Processing, Post-Translational

KW - Receptors, Cell Surface

KW - Receptors, Urokinase Plasminogen Activator

KW - Recombinant Fusion Proteins

KW - Sequence Homology, Amino Acid

KW - Skin

KW - Tetradecanoylphorbol Acetate

KW - Wound Healing

KW - Journal Article

KW - Research Support, Non-U.S. Gov't

U2 - 10.1042/BJ20031478

DO - 10.1042/BJ20031478

M3 - Journal article

C2 - 15012588

SN - 0264-6021

VL - 380

SP - 845

EP - 857

JO - Biochemical Journal

JF - Biochemical Journal

IS - Pt 3

ER -